DNA - Science topic

The oldest relevant reference in the OED is to a paper by Gamow and Yčas from 1955, namely Statistical Correlation of Protein and Ribonucleic Acid Composition.

I don't see a question, but there is a relevant observation to be made: Dealing only with genetics any black or white males are closer than any male and female. By the way, the color pigeonhole society employs are simplistic to the point of stupidity. 'Whites' are pink, not white unless they happen to be albinos. 'Blacks' are almost always some shade of brown,and Asians have a remarkable range of color tones.

Yes and no. There are minor differences which can cause your results to be false. Reducing the grid box would definitely decrease the total volume so that the interactions will increase just like in a more concentrated situation. However, when your ligand and/or receptor (target molecule) goes outside the walls of grid box, it comes back from the other side (Leaving the grid from right, entering from left etc...). So in that sense, you create a new collision probability for the atoms to interact. Let's assume you have a tiny drug that you want to bind to your large protein to inhibit/suppress its activity. In terms of theoretical chemistry, it may not have any chance to attack and residue on the right side of that protein. As you limit the grid-box, some right parts of that protein will enter from the left side and your drug will be able to interact that side as well which is not supposed to happen. So if you want to increase the concentration go ahead and do it. Please do not decrease the grid-box volume to satisfy that need since it will yield wrong outcomes.

That sort of smear-y shape can be due to a few things:

loaded too much sample

dirty gel combs

removing combs too early

So, I bet if you washed & rinsed the gel casting equipment & gel rig; used fresh electrophoresis buffer; and made sure you let the agarose completely harden before removing the combs your bands will look much better.

I don't think the sizes are necessarily different from what you have predicted, pretty sure it's just the smear/wobbly bands that's making it hard to estimate the size.

Good luck!

1. Type of Sequencing Technology: Different sequencing platforms have specific requirements and capabilities regarding DNA fragment size. For example, short-read sequencing technologies like Illumina typically require smaller DNA fragments (e.g., 150-500 base pairs), while long-read sequencing technologies such as Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) can handle larger DNA fragments (e.g., several kilobases to tens of kilobases).

2. Genomic or Targeted Sequencing: If you're performing whole-genome sequencing, the DNA size should be representative of the entire genome, so fragmentation methods may be used to generate smaller, randomly sized DNA fragments. For targeted sequencing, such as amplicon sequencing or targeted capture, the DNA size is determined by the specific regions of interest and the primer or capture probe design.

3. Desired Coverage and Resolution: The size of DNA fragments can affect sequencing coverage and resolution. Smaller fragments may provide higher coverage across the genome but may result in lower resolution for structural variations and long-range genomic interactions. Larger fragments can capture more long-range genomic information but may require specialized sequencing technologies.

4. Sample Quality and Integrity: High-quality DNA with minimal degradation is essential for successful sequencing. Fragment sizes should be selected to ensure that the DNA is intact and free from degradation, especially for long-read sequencing technologies that require longer DNA fragments.

5. Downstream Analysis Applications: Consider the downstream applications and analyses that will be performed with the sequencing data. For example, if you're interested in identifying structural variations or long-range genomic interactions, longer DNA fragments may be preferred. Conversely, if you're focusing on variant calling or SNP analysis, shorter DNA fragments may be sufficient.

6. Library Preparation Methods: The choice of DNA size may also depend on the library preparation method used for sequencing. Different library preparation kits and protocols have specific requirements for input DNA size and fragmentation methods.

we are working on colored cotton fabric dna extraction kit

Hi, Angrand, I use qPCR for detecting specific genera using genera specific primers. But detecting species would be a bit difficult. you can go with metagenome targeting whole 16S for species identification.

Thanks

Ruslan Kalendar

I added 3 M NaAc pH5 and the purification still didn't work. Still 0% recovery.

I used pH strips too. The pH is low but still 0% recovery.

Dear Colleague,

I trust you are doing well. In response to your request for suggestions on reasonably priced oligonucleotide synthesis services, both within India and internationally, I am pleased to provide a comprehensive overview aimed at facilitating your decision-making process.

When selecting an oligonucleotide synthesis service, consider the following criteria to ensure you receive the best value and quality for your research needs:

Before finalizing your decision, it may be beneficial to request quotes from multiple providers and evaluate any bulk order discounts or promotional offers that could further optimize your investment. Additionally, reaching out to your professional network for firsthand reviews and experiences can provide valuable insights into the reliability and quality of the services you are considering.

Should you have any further inquiries or require assistance in contacting these services, please feel free to reach out.

Best regards,

With this protocol list, we might find more ways to solve this problem.

Yes, infusion cloning, also known as In-Fusion cloning, is a popular method used by many researchers to insert short DNA fragments into vectors with high efficiency. In-Fusion cloning is a versatile and powerful technique that enables the direct joining of DNA fragments—such as inserts and vectors—without the need for restriction enzymes or ligases. This method relies on the homologous recombination of linearized vectors and insert DNA sequences that share complementary ends.

How It Works:

In-Fusion cloning exploits the natural homologous recombination mechanism to seamlessly join DNA fragments. To prepare for In-Fusion cloning, both the vector and the insert DNA must have complementary ends, which can be created through PCR amplification using primers that contain the necessary overlaps. The method allows for precise insertion of the DNA fragment into the vector at a specific location, making it highly useful for cloning short inserts, mutating specific sites within a plasmid, or adding tags to proteins.

Advantages:

Applications:

Considerations:

When using In-Fusion cloning for inserting short DNA into a vector, it's important to:

In-Fusion cloning has been widely adopted in molecular biology labs due to its ease of use and high efficiency, making it a go-to method for cloning and genetic manipulation projects.

l This protocol list might provide further insights to address this issue.

Dear Esteemed Colleague,

Thank you for your inquiry regarding the handling of baseline and threshold differences in quantitative Polymerase Chain Reaction (qPCR) analyses. Addressing these differences is crucial for ensuring the accuracy and reproducibility of qPCR data. Below, I provide a comprehensive and structured approach to manage these variations effectively.

1. Understanding Baseline and Threshold in qPCR:The baseline in qPCR refers to the initial cycles of the amplification process where the signal is too low to be distinguished from background noise. The threshold is set at a level where the signal begins to be significantly detected above the baseline, marking the point where exponential growth of the PCR product is first observed. Correctly setting the baseline and threshold is essential for accurate quantification of the initial amount of template DNA.

2. Baseline Adjustment:To deal with baseline differences, it is imperative to standardize the baseline setting across all runs. This can be achieved by:

3. Threshold Setting:Threshold differences can be addressed by:

4. Normalization and Controls:Normalize your qPCR data using reference genes that are stable across your samples. Including multiple reference genes for normalization can further mitigate the impact of baseline and threshold variability. Additionally, use positive and negative controls in each run to monitor the performance of the qPCR assay and to ensure consistency across different runs.

5. Replicates and Statistical Analysis:Perform technical replicates for each sample to assess the reproducibility of your results. Statistical analysis of the replicates can help in identifying outliers and in estimating the variability within your qPCR data, allowing for more accurate interpretation.

6. Software and Analysis Tools:Leverage advanced qPCR analysis software and tools that offer more sophisticated methods for baseline and threshold adjustment, including algorithms that can account for variability across different runs and samples.

In conclusion, dealing with baseline and threshold differences in qPCR requires a meticulous and standardized approach. By implementing the strategies outlined above, you can enhance the accuracy and reliability of your qPCR results, thereby facilitating more precise quantification and analysis of nucleic acid samples.

Should you require further assistance or wish to discuss more advanced strategies, please do not hesitate to contact me.

Warm regards.

Reviewing the protocols listed here may offer further guidance in addressing this issue.

A distance of 20 bp in DNA (I assume) has a length of 68 Angstrom. This should be close enough for a significant extent of FRET quenching by BHQ-1.

The behavior of FAM in this system could be surprising. It is likely to be at least partially quenched already due to interactions with the DNA, regardless of the BHQ. If the oligo is denatured, its fluorescence intensity could increase. In other words, for certain uses, the BHQ may be unnecessary.

Check the temperature The average Tm of both primers should be used as the annealing temperature in PCR. Increasing the annealing temperature can reduce non-specific reactions and The DNA sample may not be pure enough, so it may not amplify in certain strains. You can try washing and re-precipitating the sample DNA to remove contamination. Identifying microalgae, you can use 18S rDNA for eukaryotic microalgae and 16S rDNA for prokaryotic microalgae.

Hello,

Titration of mixed solutions containing nitric acid (HNO3) and sulfuric acid (H2SO4) can be challenging due to the complex chemistry involved. However, there are methods available to accurately determine the concentrations of these acids in a mixture. One commonly used technique is the "back titration" method, which involves a series of chemical reactions to determine the acid concentrations step by step.

Here is a detailed and orderly explanation of the back titration method for titrating HNO3-H2SO4 mixtures:

This back titration method allows you to determine the concentrations of both HNO3 and H2SO4 in the mixture. It is important to perform accurate measurements and titrations to obtain reliable results. Additionally, using standardized solutions and appropriate laboratory techniques is essential for the success of this titration method.

I hope this explanation helps you understand the procedure for titrating HNO3-H2SO4 mixtures. If you have any further questions or need clarification on any aspect of this method, please feel free to ask.

With this protocol list, we might find more ways to solve this problem.

Hello,

In the context of RNA extraction, both phenol:chloroform:isoamyl alcohol and plain chloroform have been traditionally used, each serving specific roles in the process of isolating high-quality RNA.

In summary, both phenol:chloroform:isoamyl alcohol and plain chloroform have roles in RNA extraction, with the choice and use depending on the specific requirements of the RNA extraction protocol and the nature of the sample. Proper handling and adherence to protocols are essential for obtaining high-quality RNA suitable for downstream applications.

Check out this protocol list; it might provide additional insights for resolving the issue.

"Help morphology", what does this mean? Belief is for religion, it is not an intellectual position nor method of science.

If a restriction enzyme is CpG methylation sensitive, you need to consider the following:

Overall, optimizing conditions and exploring alternative enzymes are key to making CpG methylation-sensitive restriction enzymes work effectively for your experiments.

l Take a look at this protocol list; it could assist in understanding and solving the problem.

The presence of genomic DNA in the sample can be considered as a contaminant because of that during running Real time PCR, it affects quantity of cDNA (which is expected to come from expressed genes/transcripts). Since some DNA probes can bind to any double stranded DNA molecules, the presence of genomic DNA can affect quantification of cDNA.

Ajay Yadav i tried to modify RNA to DNA by using discovery studio by selecting deoxyribose. However when i select Show sequence, I can still see Uracil instead of thymine. Nikhil Maroli Ajay Yadav Do you have any suggestions how to convert structure obtained by RNA composer to DNA aptamer.

Hi Harshita

lot of possibilities, but the main ones are to go to the NCBI or UCSC database (for instance, just type NCBI XXXX YYYY UTR region, where XXXX is your bacteria and YYYY your gene) in google.

or just give the species and target in research gate...maybe someone could answer ;)

all the best

fred

Have you found a way to efficiently enrich host DNA? I also encountered this problem.

Given the low quantities of linearized DNA that will be present you definitely need some form of enrichment protocol. A miniprep kit might work.

In certain DNA replication errors, particularly during template-switching events like strand slippage or misalignment, the resulting duplication can indeed be either in the same orientation (tandem duplication) or inverted (inverted duplication) relative to the original sequence. This can occur under specific circumstances and mechanisms during DNA replication.

In template-switching, which is often associated with replication errors like slipped-strand mispairing, the DNA polymerase temporarily dissociates from the template strand and switches to a nearby template strand before continuing replication. The orientation of the newly synthesised DNA strand and the template strand to which it switches can determine whether the resulting duplication is in the same or inverted orientation. Here's a basic explanation:

The specific circumstances and outcomes of template-switching events can vary, and they are influenced by factors such as the length and sequence of the repetitive elements in the DNA, the timing of the switch, and the precise mechanisms involved in DNA replication. These events can lead to genetic rearrangements and contribute to the diversity of genomic structures, which can have implications in terms of genetic variation and evolution.

Ryan Gourlie

The second question is easier, and the answer seems to be no

As for simultaneous extraction of RNA and DNA from blood, I am not aware of any convincing kits/protocols

(eg

)

and especially there is no comparison/standardization of any sort. If you can, isolate RNA/DNA separately; if not, can look at the Qiagen Allprep kits and experiment with blood or isolate total nucleic acid and treat w RNAse/DNAse.

Hello Miriam Negussu

Yes, you may use RNAse A for treating DNA which has already been extracted. You may add 50μg/ml of RNase A and incubate the mixture for 1 hour at 37 degree C. The treatment of DNA with RNase A should be done in Tris buffer at the end of the extraction protocol. Salting out step can be repeated as before according to the protocol which you have followed to obtain DNA.

The DNA pellet may be dissolved in Tris-EDTA for DNA protection from degradation by metal dependent nucleases during storage.

Best.

Manoj Patel

1) Histones are primarily known for their role in packaging and arranging DNA into nucleosomes. However, there is evidence that histones can also be found outside the nucleus, including in the cytoplasm. In the cytoplasm, histones have been shown to bind to lipid droplets to fight intracellular bacteria. Additionally, histones can shuttle between the nucleus and cytoplasm. Karyopherins, which are nuclear import receptors, can interact with histones and function as chaperones, protecting histones from non-specific interactions in the cytoplasm. Furthermore, studies have suggested that karyopherins may participate in the deposition of histones into nucleosomes. Therefore, while histones are primarily associated with nucleosomes, they can also exist as individual entities in the cytoplasm and participate in cellular processes outside of chromosomal gene regulation.

2) Histone binding to plasmids can occur in a non-specific and non-sequential manner. Nucleosome assembly on plasmids appears to be random rather than cooperative, with nucleosome formation equally likely to occur over all regions of the DNA molecule. This random assembly is supported by the observation that reconstituted plasmids lacking linker histones show no tendency towards precipitation or aggregation. Additionally, histones have been shown to bind bacterial plasmids with high affinity and form nucleosomes in vitro. The binding of histones to plasmids can limit the access of transcription factors to their recognition sites, affecting transgene expression. Therefore, histone binding to plasmids is not specific or sequential, and it can impact gene expression by modulating access to the DNA sequence.

Upper gel:

In samples 3 & 4 there is obviously no or very little DNA.

In my opinion all other DNAs are not properly dissolved and the gel is overloaded; and you might have RNA contamination.

Ensure your DNAs are completely dissolved and as suggested by others determine the concentration and reload equal amounts.

And, as also suggested by others, you should always seek advice from your supervisor or a senior lab member.

I use ethidium bromure (BET) to visualize PCR products. This dye migrate in opposite direction of DNA. If my gel run for a too long time the dye risk to be over of small sizes. I supose it is your case.

What you can try: increase agarose concentration at 2% or 3%.

Reduce the time for the migation : 15 min should be enough. You can adjust the voltage.

You can increase your SBYR amount.

Another tip : don't use detergent to clean your gel tank

I looked into the paper from 1988 describing the method. "Thus, it is clear that alkaline conditions will permit a more sensitive detection of DNA damage, including single- and double-stranded DNA breaks and alkali-labile regions, such as apurinic and apyrimidinic sites [9] and phosphotriesters [10]"

Well, not identifying only breaks but also...

I know that people in the neighboring DNA repair group were dealing with the repair of abasic sites.

Are you using SYBR green? Anything that would show up in an electrophoresis should also show up on the real time results.

This isn't surprising at all. The template DNA + primers + dNTPs should be the same "amount of nucleic acids" at the start and end of PCR.

ATL is a lysis buffer which has SDS and EDTA in it and is not recommended for long term storage.

AL has guanidine hydrochloride and malic acid to denature proteins.

TE is Tris/EDTA so uses tris to maintain the pH and EDTA to chelate metal ions which would damage the DNA.

A crude method for getting some DNA for PCR might simply be to boil the cards in some distilled water for 15 mins.

This research gate question covered the functions of the buffers (https://www.researchgate.net/post/What-is-the-exact-composition-of-AL-buffer-of-DNA-isolation-kits#:~:text=For%20buffer%20AL%2C%20it%20says,of%20the%20buffer%20they%20use.)

I agree that and increased 230 is usually due to salts and has a bad effect on the ratios. I think that ethanol is better than isopropanol because ethanol tends to preciptate fewer salts so all I can suggest is to check the rnase buffer for SDS and if it contains sds then do not add the buffer just run the rnase as Rohit Kumar suggests. Also check your ethanol volumes very carefully to avoid using too much ethanol. So If your dna volume is very small it may be more accurate to increase the dna volume so that adding the alcohol is very accurate...eg 5ul dna plus 10ul ethanol is prone to pipetting error but 50ul plus 100ul the accuracy of the pipetting should not be an issue. If the amounts of dna are very small then you may want to add a coprecipitant like glycogen or linear acrylamide to increase the precipitation yield of the dna.

It may be worth using your pipettes to weigh a few volumes of water to check that the pipette used for measuring the dna volume is not reading low and the pipette used to add the ethanol is not reading high

DEL which entails a collection of small molecules covalently linked to DNA that has unique information about the identity and the structure of each library member.

In future, promising job opportunities will be beconing on those professionals who are clinically savvy especially in the pharmaceutical/drug and biomedical sciences. More human capital will be needed in numerous drug discovery programmes in future.

Hi Moises , I agree with Jatesh , yes you can use EB without any EDTA

In my opinion, kit tubes should be fine. If adherence is the problem, you can short-spin it after adequate tap mixing.

Hope, it helps.

Dear Pankaj Yadav

As part of my ongoing research within the Energy-Impulse-Information-Tensor (EIIT) framework, I am delving into the intricate characteristics that distinguish living and non-living systems, as well as the profound concepts of life and death. This framework offers a holistic perspective to comprehend these concepts, viewing life as a complex system that harnesses energy, responds to impulses, processes information, and evolves within the fabric of spacetime.

1. **Energy**: Living organisms actively harness and convert energy to carry out various functions such as growth, reproduction, and maintenance. This energy is typically derived from food (in animals) or sunlight (in plants) and is used to drive biochemical reactions. In the EIIT framework, this energy utilization can be represented as a flow of energy within the tensor, which is absent in non-living entities. Non-living entities, like a rock or a molecule of water, may contain potential energy or kinetic energy due to their physical state, but they do not actively convert or use energy for processes like growth or reproduction.

2. **Impulse**: Living organisms respond to stimuli or impulses from their environment. This could range from a plant bending towards light to a human responding to a complex thought. In the EIIT framework, these responses can be represented as changes in the impulse components of the tensor. Non-living entities, on the other hand, do not respond to impulses in the same way. They may change state due to external forces, but these changes are not active responses and do not involve information processing.

3. **Information**: Living organisms process information. This is evident in the genetic information carried in DNA, which guides the growth and development of an organism. Additionally, organisms process information from their environment to make decisions. In the EIIT framework, this information processing can be represented as changes in the information components of the tensor. Non-living entities do not process information in the same way.

4. **Tensor**: The tensor in the EIIT framework represents the spacetime in which the living organisms exist and evolve. They are born, they grow, they reproduce, and they die. Over generations, populations of organisms evolve, driven by the forces of natural selection and genetic drift. Non-living entities, while existing within spacetime, do not evolve in the same way.

The boundary between living and non-living can be blurry, especially when we consider entities like viruses. Viruses exhibit some but not all characteristics of life. They contain genetic information and can evolve over time, but they do not have their own metabolism and cannot reproduce without a host cell. In the EIIT framework, viruses could be considered as systems that can process information and evolve over time, but do not independently utilize energy or respond to impulses.

As for the difference between life and death, in the EIIT framework, death could be considered as a state where the organism no longer processes energy, responds to impulses, or processes information. The system that was once a living organism no longer evolves over time in the same way. However, it's important to note that while the individual organism ceases to function, the energy and matter it was composed of continue to exist and transition into other forms and systems. This aligns with the principle of conservation of energy and the concept of information conservation, suggesting that while the specific configuration of energy and information that represented the living organism is gone, the energy and information themselves persist.

Regards,

Alessandro Rizzo

I am not a biologist, but I should think that hidden markov models comes out as a prerequisite to undertake this kind of tasks.DNA strands for various genes are aligned along a given strand sequence--the DNA of your mouth has a unique code, different from that of your legs. the mathematics which models this kind of sequence is operations research and biostatistics. to build these models one assumes/ presupposes that you are familiar with what stationary markov chains are, and that you are a bit familiar with bayesian statistics. This will give u added advantages u read and make efforts at understanding & building such sequence-based models.

a link such as the one presented hereunder for your perusal gives you a headstart:

OK, given the context of the research, you are going to want to start by using regular PCR to see if the region of the gDNA can be amplified at all.

In-silico predictions often have errors. You'll save yourself a LOT of stress if you can show that the genomic region exists in the DNA.

Besides, you'll need to clone that region of the genome to make a standard curve for your qPCR. It's a "win win" to check for the gDNA first.

There is no specific reference available that discusses the issues with extraction of eDNA using ZymoBIOMICS DNA/RNA Mini Kit from roots. However, there are several studies that have evaluated different DNA extraction methods for eDNA analysis from various environmental samples, including water, soil, and human milk. These studies have used different DNA extraction kits and protocols and have reported variations in DNA yield, quality, and taxonomic composition. For example, [10] found that filtration through a 0.2-0.6 μm pore size PC filter, followed by extraction with MoBio PowerSoil DNA Isolation Kit was optimal for quantification of eDNA from fish. [11] reported that eDNA collection by filtration and subsequent extraction/purification using a DNeasy Blood and Tissue DNA extraction kit (Qiagen) or PowerWater DNA Extraction Kit (Qiagen) is the most common procedure for detection of aquatic macroorganisms. Ojo-Okunola et al. (2020) found statistically significant differences in taxa prevalence from DNA extracted using ZymoBIOMICS™ DNA Miniprep kits compared to other kits for human milk bacterial profiling. [12] tested nine different DNA extraction methods using three commercial kits and compared them in terms of DNA extraction ability from human fecal sample. [13] proposed an optimized protocol for eDNA extraction from oligotrophic and degraded water samples using a specific kit. [14] evaluated the performance of four commonly used DNA extraction kits, including ZymoBIOMICS DNA Miniprep Kit, for studying cervical microbiota. [15] reported that different DNA extraction methods may cause changes in yield and stability, resulting in an inaccurate estimation of eDNA. [16] compared different methods for extraction and purification of environmental DNA from soil and sludge samples and focused on methods that are appropriate for the extraction of eDNA from very limited sample sizes (0.1 g) to enable a highly parallel approach. Therefore, it is important to carefully select the appropriate DNA extraction method and kit based on the sample type, target organism, and downstream analysis.

Did you manage to fix this issue? How? I am experiencing this problem, too. Except I ethanol precipitate my amplified DNA before resuspending it in water REALLY WELL and running it on the gel. The samples just hang around in the wells and do not migrate down.

Believe the data. If the sequence data is good quality and it is matching up with something other than what you expect, you should believe that data but figure out why. It could be the organism you think you are working on is not really that organism but is something else. Or it could be that your culture is contaminated.

You might need to provide more experimental design information.

Are you dealing with very low expected [template], such that Cqs are often 30+ (in which case "no amplification" genuinely implies "no target"), or are you dealing with fairly robust [template], i.e. Cqs of 20-30 (in which case "no amplification" means "freak weird event")?

If the former, the extent of quantitative info you can glean is going to be more limited anyway (stochastic template numbers will be inherently variable), and you should flag the well as "no amplification".

If the latter, you just ignore it.

Pure ethanol will evaporate if you leave the tube open. It does take a while though.

Dear friend Grigorii Rudakov

The choice of the number of nucleotides in a sticky end depends on several factors, including the size and complexity of the DNA molecule, the stability of the hybridized DNA, and the specificity of the interaction between the complementary sticky ends.

While it is possible to use three nucleotides for a sticky end, it may not be the most common choice because it can result in a weaker interaction compared to using two or more nucleotides. This is because a longer sticky end with more nucleotides provides a greater number of hydrogen bonds between the complementary base pairs, resulting in a stronger binding affinity.

Additionally, the length of the sticky end can affect the specificity of the interaction. Using a longer sticky end with more nucleotides can increase the likelihood of non-specific interactions between complementary sequences, leading to off-target binding and reduced efficiency in constructing the desired structure.

In your case, using a three-nucleotide sticky end may be sufficient for your specific design, but it is important to consider the potential trade-offs between specificity and binding strength. You may also want to consider using other strategies to increase the stability and specificity of the interaction, such as incorporating modified nucleotides or optimizing the sequence of the sticky ends.

Is it usual to amplify DNA with thiolated Primer? Otherwise, you may need to check how thiolated primer affects PCR. I am not sure but thiolated primers might form di-thiol between themselves.

The immobilizing DNA amount might not be enough ( I did not check it).

During incubation of your DNA on the gold surface, it should not be dried. TCEP makes the solution acidic. I recommend you need to mix TCEP and DNA in a proportionate buffer. Usually, I used PB as the article. I am not sure that such long incubation. Anyhow, if you add salt such as NaCl to the incubating solution. It helps to increase the immobilized density of DNA because It reduces electrostatic repulsion between DNA. You might refer to these two articles and 10.3390/bios10050045.

Good luck

Hi Yun,

Generally, when using the whole slide, scraping is done after rehydration (post-xylene etc.). However, based on your question it looks like you do not want the whole section.

Right at the beginning before deparaffinization, you could use a very sharp blade and then cut around the area you want. Don't scrape it or anything and leave the area still attached. Make sure you make a good cut through the tissue. This would the best time to make the cut when the slide is untouched and section is firm. Then you can do deparaffinization with xylene etc. and when you finish, the rehydration step, it will be easier to just remove that area by scraping, or use a regular sharp shaving blade to "lift" off the area.

Trying to cut after the rehydration step you will definitely be dealing with more fragile tissue and cutting may not be that easy. Don't do too much baking etc. this will jsut degrade the RNA/DNA that much more.

Hope this helps!

Good luck!

Hi , in selection of mismatches (SNPs) it easily works. Coupling flourcent dyes to such primers can convert PCR to RT PCR .

Just curious, did you get good protein expression using beta globin gene UTRs instead of the genes specific UTR? I am thinking of using alpha or beta globin UTR too. Dolon Maji

Dear Edeildo Silva-junior,

Could you write, please, which software you can recommend for docking including metal ions? Thank you very much in advance.

Thanks Adam, very useful. Not sure how I missed this one in my searching.

I would use a 1% agarose gel . Heat your ladder to 89c and allow to cool. The fragments sometimes reanneal to give better bands. I do not like the situation where the ladder runs slower than the product so borrow another ladder and run it as well in case your ladder has too much salt added and is running slowly. If the ladder is running well then all of your bands are the wrong size and too small so the bands may even be primer dimer. Run a pcr again but include one sample that has NO added dna and see if these sample generate a band in which case you either have contamination amplifying or you have a primer dimer in which case use a hot start Taq and use much less primer

If your dna is already in solution then spinning down will move all of the wetting on the tube sides into the solution at the bottom which will minimise the loss of water to evaporation in storage although small volumes will evaporate even when stored in a freezer.

Your dna will remain in solution during spinning and storage because addition of salt and alcohol is needed for dna precipitation. Once your dna is dissolved it should stay dissolved even in freezing and thawing

I have heard, but do not remember the source, that 100% alcohol/absolute alcohol can be used for DNA studies. Normally 70% alcohol is used for storage in alcohol. Some recommend 70% with 5% glycerol for long-term storage. If possible, freezing, as mentioned, is best for DNA studies. The colder the better. -18 degrees C, is standard in household freezers. Dry ice holds - 78^o and can be used for short-term freezing. Liquid nitrogen is even colder, but requires special equipment.

Dear Emilie,

The solution may be very simple: if you clone in Lambda DNA digested with PstI into PstI-digested pU19, and then digest all of the plasmid DNA samples produced from cultures inoculated with blue or white colonies and selected with ampicillin, then all of your samples will include a band at 2.7 kb corresponding to ... pUC19 ... which is the source of the ampicillin resistance and will turn the colonies blue if there is no insert of DNA at the PstI site.

Your gel images suggests that a number of your samples contain Lambda DNA inserts, but since the band intensities vary a lot, I'd be a little careful in interpreting some lanes with bands above the pUC19 band, as this may represent partially-digested plasmid DNA. Lanes 3, 6, 7, 9, 10, 13, 15 and 16 look interesting. You could look at a Lambda restriction map to see the range of PstI fragments it would produce, and map them onto the fragments you can see in your gel.

Regards, Andrew

My question will be, why do you have such a strong smear below your DNA bands?

Is this a plasmid?

Looks like your samples are contaminated with DNAase.

Depending on the host bacteria and extraction kit you might need to do a washing step.

First, I'd re-state the question to assure that I understood it correctly. A nucleotide sequence of length l contains a palindrom with unit of length k. The palindrom is not exact; there can be from kmin to k matches between units. The distance between palindrom units can be from smin to smax. First and last sub-sequences of length k are not exact matches of any palindrom unit.

My solution. Let's omit the last condition for now. How we search for a palindrom with unit of length k? Take any subsequence of length k and search for a 'match'. Searching for a 'match' is equal to checking (l-k-smin) subsequences, because the unit itself occupies k nucleotides and a spacer can't be shorter than smin nucleotides. In each window the probability of hit is (1/4)^(kmin), if every nucleotide has equal probability of occurrence. The probability of having 1 or more hits then is equal to binomial cdf with the number of attempts equal to n-k-smin, the probability of success equal to (0.25)^kmin and number of successes equal to 1. For example, GSL function gsl_cdf_binom_Q(n-k-smin,0.25^kmin,0) would give the answer. The last paramerter is zero, because the function computes the probability of more than x successes, i.e. 1 and more in this case.

Now, let's include the last condition. It is important to define what 'does not match' mean. I suppose that it means that we can't find the second palindrom unit at postions 1 and l-k. So, the number of windows that we check has to be decreased by 2. The final answer would be:

F(n-k-smin-2,0.25^kmin,0), where F - binomial cdf.

For varying length the answer would be a weighted sum of those propabilities, with weights equal to the probability of observing given legnth. So, if all lengths have equal probability, this is the mean.

I checked the answer on a synthetic set and it seems it is correct or close to being so.

Hello all,

Thank you so much for your answer. Lately I have tried DNeasy PowerClean Pro Cleanup Kit for an additional clean up process of DNA samples and ran PCR. I think I saw some bands in the region of 200-300bp.

It is just coincidental

First you should specify if your are interested in a particular gene, its gene expression, or just genomic DNA. If you are interested in a gene or a specific genomic or plasmid region, then qPCR is a good method. Just design primers for that, and for a control gene or region, and build a standard curve with known concentrations or copy numbers to extrapolate fluorescence intensity values

If you have a plasmid or PCR product, you can estimate the concentration by comparing your sample to a known DNA ladder. The product insert will tell you the specifics for the ladder. ImageJ could make it a bit more quantitative, but you still need samples with a known concentration as a reference. Also, know that ethidium bromide (or SYBR Safe or whatever stain you use) will have an upper saturation limit.

No one every bothers to quantify a DNA extraction from eukaryotes on an agarose gel. All you would ever see is a faint smear. If you really need to know the concentration of genomic DNA, ask to borrow a spec or nano drop. The chemistry department should have one.

You will get a rough estimate of the "quality" of your RNA by looking for 2 bright rRNA bands + a faint tRNA smear on an agarose gel. In fact, it's best practice to use an agarose gel rather than a spec since the spec won't tell you if the RNA is degraded.

Good luck!

Sure Azka Saleem

Steps u can consider following.

1. Download the SGD data as a fasta file/if separated as many files, u can merge all into single fasta file.

2. Make a list of IDs in MS excel whose sequences you need to extract.

3. Install TB tool in your dekstop, and search for fasta extract option, where u need to provide the database fasta file and the IDs and click ok.

4. In the output file you will have your required IDs sequence data.

If you still find it difficult to perform, u can mail me in [email protected]

DOI: 10.1039/b804675c

This paper explains very well how unfolding and melting of DNA hairpin works. kindly have a look.

Hello Lanka Sai Meghana

The process or act of exchanges of genes between chromosomes, resulting in a different genetic combination and ultimately to the formation of unique gametes with chromosomes that are different from those in parents is what is called genetic recombination. In this process, there is rearrangement of DNA sequences by the breakage and re-joining of chromosomes or chromosome segments.

Two very close-together genes will have very few recombination events and be tightly linked, while two genes that are slightly further apart will have more recombination events and be less tightly linked.

So, the linkage is indirectly proportional to the distance between genes, the less the distance between the genes, the less is the cross over frequency between them and the more is the linkage frequency between them.

Best.

hi Jessica

you can also use chromas which is worlwidely used (http://technelysium.com.au/wp/chromas/).

all the best

fred

Don't use this website to ask for help on your homework.

This is very good and relevant question. I think same with extraction procedure tissues of animals.

I have similar question. we would like to buy spectrophotometer for plant phyisology lab. what is your opinion about Thermo Scientific

GENESYS Vis and UV-Vis Spectrophotometers? or any other suggestion?

to measure chlorophyll, some enzyme, protein and ...

How it being calculated attached file please?

One of the issues that is holding the concept of an RNA world back from being more scientifically useful - irrespective of whether there ever was such a thing - is that we don't use the idea in the scientific way it was intended. Just like any other prebiotic scenario, it is not (nor has it ever been) a scientific hypothesis. In fact, scenarios are usually not intended as such. Scenario authors from all niches (including RNA world) have pointed out that scenarios themselves are untestable. However, they guide thinking and allow to conceive of hypotheses that are testable. If we go through the old literature we find very explicit passages to support this fact.

For the specific authors advanced in the question, G.F. Joyce and L.E. Orgel, we have a passage from 1999 in "prospects for understanding the origins of the RNA world". (The RNA World 2nd ed. 49-77).

"The presumed RNA World should be viewed as a milestone, a plateau in the early history of life on earth. So too, the concept of an RNA World has been a milestone in the scientific study of life's origins. Although this concept does not explain how life originated, it has helped to guide scientific thinking and has served to focus experimental efforts."

You can find this point of view expressed in foundational work for all niches related to the popular scenarios today. But you can also find it for scenarios most people in origins have never heard of. E.g. the idea that celllular life started with terpenoids found in G. Ourisson and Y. Nakatomi's "the terpenoid theory of the origin of cellular life: the evolution of terpenoids to cholesterol. (1994) Chem & Biol. 1 11-23".

"The hypothesis provides an attractive way of ordering the terpenoids: like all evolutionary theories, it cannot be tested directly. The ideas summarized here do, however, suggest a multitude of experiments having some bearing on the fundamental and fascinating question: how did the first cells appear? We hope to carry out some of them."

A related line of thought - but highly influential - is the exposition by Harold J. Morowitz from 1992 in his book "Beginnings of Cellular Life: Metabolism Recapitulates Biogenesis". If we go to the conclusion, we find this explicit clarification on the distinction between a genuine scientific theory and a scenario:

"at this stage of the thought process, it is important to focus on the hypothesis that intermediary metabolism recapitulates prebiotic chemical evolution. This hypothesis is not a strictly vulnerable theory in the Popperian sense, but it does provide us with a valuable heuristic method for using modern knowledge of biochemistry to search for events that have left their trace. If the intermediary metabolism of autotrophs does not recapitulate biogenesis, then the discontinuities will have to be explained."

More than 2 decades back, many authors made a clear distinction regarding this nuance. Scenarios are here to help: they guide thinking and design experiments. They only guide thinking in a scientifically meaningful direction as long as we can easily abondon scenarios and enthusiastically continue replacing them with new, more informed scenarios. A situation where a scenario gets entrenched and where researchers treat it as a scientific hypothesis is - by construction - hard to escape.

In fact, this is exactly the situation that many researchers have described around the 80s, when criticism mounted against the prebiotic broth scenario. The passage from Wächtershäuser's 1988 "Theory of a Surface Metabolism" is telling:

"The prebiotic broth theory has received devastating criticism for being logically paradoxical (11, 135), incompatible with thermodynamics (11, 144, 160), chemically and geochemically implausible (134, 136, 144), discontinuous with biology and biochemistry (160), and experimentally refuted (135, 160). The reason for the tenacity with which it is retained as accepted dogma has been forcefully and clearly stated by Scherer (126): "If this rejection is substantiated, there will remain no scientifically valid model of the selforganization of the first living cells on earth."

Clearly, the broth scenario had overstayed its welcome. One reason for this is that its 'claims' (which for a scenario can only be speculations) were too much in contradiction with claims from fields of science that do not suffer the same restrictions when it comes to testing and refuting their theories. One example of a very controversial idea that can be found in Haldane's formulation of a broth scenario, is the purported necessity of a long, highly functional protein randomly emerging from a soup, as an extremely rare event: we expect this to be prohibitively unlikely and hence a far from parsimonious explanation.

Quite a few of the critiques voiced against the prebiotic broth scenario are equally valid critiques of some scenarios we have today, including RNA world.

The RNA world is an old and multifaceted concept. There are contrasting formulations that make different claims (to be interpreted as speculations) about history. As with the prebiotic broth scenario (and any scenario), it has raised genuine scientific objections. These have remained largely unadressed, in spite of its long dominance.

It is instructive to bear in mind that scenarios don't come from nowhere. They're fairly detailed speculations about purported historical events. To make them, each author makes assumptions. Some of these concern speculations that later became testable, e.g. about chemistry and physics. You will find different scenario authors make different assumptions and different arguments (and flaws therein). There's an inevitable bias here with respect to the fields an author is trained in. Some of the foundational assumptions in popular scenarios like RNA world are at least 50 years old, but some unchallenged assumptions date back to a literature that is more than a century old. A time before IUPAC, modern quantum mechanics, genetics, and so forth.

That has been enough time to forget that scenarios like RNA world are by construction not testable hypotheses and that they were not intended as such. Scenarios are here to guide thinking, to inspire experiments. The best thing a scenario can do for us, is generate insights that spur us to change the way we think and thereby necessitate replacing our old scenarios with new ones, and repeat the cycle. The science coming out of the community today is a lot more conducive to doing that than previously.

The same cannot be said for the rather myopic RNA-centric framing of a question in the cited passage, which attempts to elevate RNA world to more than a scenario. Rather than forcing ourselves to think about the rather narrow and outdated proposal by Joyce and Robertson, ("consider the alternative possibility that RNA was preceded by some other replicating, evolving molecule"), it is more productive to critically revisit all the things that have been assumed and argued when the concept of an RNA world was conceived and how which of these premises are considered valid or plausible today, and which ones back then. Is there a formulation of RNA world for abiogenesis that is logically sufficient? And if so is it logically necessary that abiogenesis proceeded this way?

It is also instructive to check how much of the logic was sound. e.g. the rhetorical tricks employed in RNA world introduce all sorts of hidden assumptionsm.

As an example of the latter: some still justify an RNA world by the party trick 'chicken-and-egg' question 'protein or RNA, which came first?', only to conclude with 'RNA, it encodes proteins' and hastily conclude with an even stronger 'RNA-first' for abiogenesis. 'chicken-and-egg' fallacies are nothing new in origins. In fact, they were already identified as such long ago. E.g. in chapter 8 of "Seven Clues to the Origin of Life (1985)" by Cairns-Smith, there's an illustrated passage detailing that these types of paradoxes in origins frame the question in a manner that prevent us from considering scaffolds.

"

The fact is that even the so-called simple organisms such as E. coli are very complex enterprises with all sorts of things going on together. There is plenty of scope for accidental discoveries of effective new combinations of subsystems. It seems inevitable that every so often an older way of doing things will be displaced by a newer way that depends on a new set of subsystems. It is then that seemingly paradoxical collaborations may come about.

To see how, consider this very simplified model - an arch of stones: This might seem to be a paradoxical structure if you had been told that it arose from a succession of small modifications, that it had been built one stone at a time.

scaffolds that starts like this:

This might seem to be a paradoxical structure if you had been told that it arose from a succession of small modifications, that it had been built one stone at a time. How can you build any kind of arch gradually? The answer is with a supporting scaffolding. In this case you might have used a scaffolding of stones. First you would build a wall, one stone at a time:

Then you would remove stones to leave the 'paradoxical' structure.

"

It should be noted that in 2022, even in RNA-world, very few scholars remain that find RNA-first a convincing idea. As a scenario, however, it is not useless: it is instructive to consider what the underlying ideas are that at some point in time made such a highly specific idea compelling to so many of us.

A fixed motif in scenario papers is to start explicitly and implicitly assuming a few things about what chemistry can and cannot do and some properties of abiogenesis. These sort of assumptions used to be spelled out routinely, also outside scenario papers. Let me give two examples.

The original 1953 paper for the "Frank Model" "on spontaneous asymmetric synthesis", has the passages

".. the defining property of a living entity the ability to reproduce its own kind ...

confining attention to chemical molecules, the complexity of any having this essential property of life is likely to be great enough to make it highly improbable that it has a centre of symmetry."

(*I should point out that Frank makes an important error here: the capacity for molecular reproduction is not a molecular property but a property of a reaction network. If we add an additional thermodynamic criterion this property is autocatalysis and we can then check this claim from the IUPAC definition: https://goldbook.iupac.org/terms/view/C00876. It turns out there are trivial ways to make small networks that have this property https://chemrxiv.org/engage/api-gateway/chemrxiv/assets/orp/resource/item/60c74d67469df42226f44295/original/emergent-autocat-animation.gif.)

The point to retain here is that Frank considers it to be generally accepted that one can assume this property to be prohibitively rare in chemistry. This belief was wideheld, and we can e.g. read in "the units of selection" (1970) by Lewontin a summary on scientific views on abiogenesis

"The present view ... Since there was no autocatalysis, there was no reproduction or heredity and so no possibility of natural selection."

The coacervates in Oparins scenario were notably invoked to adress this issue.

When it comes to assumptions in scenarios, this systematically involved conjecturing that chemistry 'in the wild' intrinsically and deterministically becomes a 'mess', undergoing no meaningful complexification, and for which no reproduction and evolution can reasonably be expected. From there, it appears that no process of abiogenesis should conceivably occur naturally, and thereafter some specific sequence of exceptional events is proposed as plausible, because it appears to be the sole contender.

Let us make more explicit why this is not an innocent procedure:

We still find our understanding of 'basic chemistry' to be plagued with limitations and long-lived misinterpretations (e.g. 2 days ago we learned that methyl substitution destabilizes radicals instead of the textbook knowledge that it stabilizes them ).

Moving beyond the basics, we by and large lack a lot of formal theory, experiment, or even a simple reference frame for the things that happen then. Joyce and Robertson honor the tradition of purporting from the outset that 'chemistry in the wild' becomes an intractable mess. The issue is that we don't know at all if that's the case. We cannot assume this from the outset, we need to extensively study it. We require extensive experiments and theory and a reference frame for all the phenomenology associatied with complex systems (e.g. multiple components, compartments, multiple forms of nonequilibrium driving, length scales, time scales).

In making the routine assumption of 'messy, intractable chemistry that can neither complexify nor multiply', we have decided in advance that, once we finally understand 'chemistry in the wild' with its 'so-called intractible mixtures', it cannot have any bearing on abiogenesis. Let alone explain it.

That is a disproportionately bold conjecture about fundamental science, and a very consequential one: all historical scenarios - RNA world being one out of many - have been justified by formulating conjectures of this sort (many authors also insist on other properties, e.g. chemistry being deterministic). Clearly, it should be the first priority of everyone in the field to test this conjecture, by extensively and rigorously studying complex chemical systems as an end in itself. If the conjecture is correct, it provides an important validation for historical scenario approaches. If the conjecture turn out wrong, we are in a much better position to conceive of more scientifically informed scenarios, but potentially the approach will change entirely.

In presenting it as such, I am making it appear as if it could be an open question whether the chemical conjectures underpinning our scenarios in origins may be true or not. In fact, we have learned quite a few things in the meantime. And some clumsy mistakes were made elsewhere.

- Determinism:

When it comes to chemistry being deterministic (a key tennet of e.g. Sutherlands scenario and Wächtershäusers surface metabolism): upon critical evaluation of what is known of basic chemistry this idea becomes unacceptable, especially when considering the chemical processes on the surface of a planet, as opposed to a quick reaction in pyrex.

1) insofar as it is reproducible, modern chemistry owes much of it to big strides of standardization in glassware, methodology, synthesis protocols (e.g. usage of stirring bars).

2) lab chemistry exhibits many forms of contingency. This is particularly the case when it comes to phase behavior, e.g. habit modification, polymorphism. Aspirin purportedly has 8 reported polymorphs, phenobarbitone 13.

3) glassware is cleaned between reactions, thereby making successive reactions in the same glassware independent. In nature, this property of independence is absent. In fact, effort to make an evolutionary classification of minerals are rooted in the opposite: that certain minerals start to form conditional on the presence of certain others. (https://pubs.geoscienceworld.org/msa/ammin/article/104/6/810/570840/An-evolutionary-system-of-mineralogy-Proposal-for)

- Autocatalysis:

A first issue to get out of the way is the misconception that autocatalysis is prohibitively rare. A prominent PI in origins (RNA world, not a chemist) told me that chemists throughout history have found exactly one example. Claims about the contents of a literature one cannot realistically have read in a lifetime is a common error we can find in the origins literature. Below are some reviews.

I should stress that these reviews discuss examples from a few niches in chemistry. These reviews do at least allow to have 100s of counterexamples to dubious claims about no autocatalysis in chemistry, but it's only a small fraction. Virtually all branches of chemistry have regular reports of autocatalysis, but very few focus on autocatalysis in its own right. And hence most branches do not review their reported examples.

By critically examining the IUPAC definitions, one can show that autocatalysis is dramatially more widespread than long thought. In part, this is because the definition applies to a wealth of situations where the term is not routinely employed. By examinging the requirements of autocatalysis as an emergent network property, one can demonstrate that this property emerges particularly readily in a heterogeneous / multicompartment context. With the disclaimer that I'm an author I refer to the following:

- Messy chemistry:

Refreshing counterexamples are afforded by the literature on systems chemistry and dynamic combinatorial libraries.

In the context of origins, a recent work that is greatly aiding in fixing our misconceptions is : https://www.nature.com/articles/s41557-022-00956-7

Where a reaction of purported immense complexity is found to exhibit highly reproducible and ordered behavior as function of environmental inputs. How chemistry exactly works on this level is still poorly understood. I think I do, but it'll have to await peer review. But we cannot in good scientific conscience take for granted anymore that chemistry becomes messy and intractable. When we do the experiments, we see something very different.

in conclusion, I want to come back to the final point of the question

No. The sientific community should strive to do what it can justify scientifically. Those that find it fruitful to relegate the RNA world - which is not a hypothesis - are justified in doing so. Notably because it is is founded on scientifically refuted premises and logical errors.

Those that find ways to make it fruitful to keep it are justified in doing so: it's a scenario, one can draw inspiration from it. Perhaps a thoroughly altered version can be developed that fixes previous issues.

Above all else, RNA is an amazing molecule that has been used for fundamental research that concerns everyone in origins, and will continue to do so irrespective of how serious the RNA world scenario is still taken.

What the origins of life community needs, first and foremost, however, is concern itself with more important matters.

Complex chemistry needs to be studied thoroughly on an experimental and theoretical level.

New scenarios are needed. And these scenarios should no longer require chemistry to have properties it doesn't have, and vice versa. These scenarios should also explicitly be appraciated for what they are, an for what they're not. They're here to help, to guide thinking, inspire experiments, produce testable predictions, update our beliefs. They are not scientific hypotheses in and of themselves.