Cloning - Science topic

I am using ImmunoCult™ Human CD3/CD28 T Cell Activator (Semcell, Catalog #10971) to activate PBMC and expand T cell. but the T cell activator contains an anti-CD3 antibody clone that fully or partially blocks all anti-CD3 antibody clones used to assess CD3 expression by flow cytometry.

Are there any other ways to avoid the interference of CD3 in the CD3/CD28 T Cell Activator it I have to gate CD3 by FACS? Is it possible to avoid this if I use a different clone No. of CD3 for FACS analysis?

Some journals seems to be fake..cloned

Hi im on my final project for my thesis and i get stucked in cloning phase. My goals is to inserting one gene to vector who has another gene too. So i have a gene called llm2-mutan and llm1 who's completely ligated with pet28a(+) as my vector/backbone. I'm trying everything like ligating in 16 C overnight, 22 C 3h, etc. My gene llm2-Mutan was a pcr product. Everytime i checking my colony using PCR with electrophoresis is always showing 1100 bp, but my goals is 2100 bp because i must insert 2 gene in one vector. I hope someone can helping me with my issues. Thank u

I have been using NEB Hifi Gibson assembly for a couple years now and I've been quite happy with it. I regularly make plasmid constructs with 4-8 fragments, and always >1/4 of the colonies are "perfect," while the remaining ones may have some SNPs at the joining sites, or be misassembled due to a repetitive region.

Some colleagues said that Golden Gate has even higher efficiency. That even with 8 fragments, it is normal for 50-100% of colonies to be perfect. Is that true? Is Golden Gate that perfect?

Hi. I successfully cloned an insert into a plasmid vector and subsequently transformed it into E. coli. However, upon sequencing, I discovered that the insert-plasmid sequence is repeated two times, making insert-plasmid concatemer.

Do you have any opinion about this issue?

Thank you in advance

I want to generate knockout cell lines (of C2C12) with the crispr/cas method. Then use FACS to get single cells in a 96-wells plate. After cell sorting I want to expand and clone the single cells, but I don't have a protocol for single cell clonal expansion. Especially, because C2C12 cells are hard to culture as single cells (from what I know from literature). Can anyone help me or please give me some advice?

Kind regards,

Floris

I am currently trying to generate expression constructs of truncated proteins for x-ray crystallography. It is my first time doing cloning. I have had success so far in PCR amplifying my inserts using primers with NdeI, Bam HI, XhoI, and EagI restristion enzyme sites. My amplified inserts are running at the correct molecular weight as confirmed by agarose gel. I always ethanol precipitate my PCR produce overnight and resuspend the DNA pellets in 10mM Tris pH 8.0 and then do a double digest of the insert using NEB's restriction enzyme cloning tool. I typically digest my inserts for ~4-6 hours and then ethanol precipitate the digestion reaction. At the same time I do a double digest my pet28 or pet 15 vector with the same restricion enzymes under similar conditions, I have checked that each enzyme has linearized my plasmid on a gel before I add the other enzyme and then combine the digestion reactions. The uncut plasmid migrates slower than the digested plasmid and the digested plasmid runs at its expected molecular weight. I also ethanol precipitate my vector after the digestion. Finally, I use T4 DNA ligase from NEB and perform the ligation reaction. I then transform 200uL of competent cells with 10uL of the ligation reaction usually. I have tested my comp cells and used a control plasmid and was able to get good transformation efficiency with only 1ng of DNA and 50uL of comp cells but since the ligation reactions tend to give little colonies I have scaled up the transformation. I mini-prep some colonies from the transformation and have done double digests for over 100 colonies now and I do not ever see a band corresponding to my insert being there. Lately I have been doing a single digest to see if I can see the plasmid molecular weight increase as a result of the insert being there, but I do not think I see an insert also. For my most recent ligation I did a 7:1 molar ration of insert to vector in a 20uL reaction with ~20ng of vector. I have also tried 50ng of vector and 20:1 10:1, 1.2:1 ratios and still i get no insert. My most recent transformation for example gave 3 colonies for my insert reaction, 0 colonies for my control of no insert +ligase, and 5 colonies for my control reaction with no insert or ligase. I haven't digested the mini-preps yet but I feel like I will not see an insert again. I would say these results have been typical for my ligations, sometimes I get ~7-27 colonies for my reaction with the insert and 0 colonies for both of my no insert controls. When I test the colonies though, I do not see an insert or shift in molecular weight indicating the insert is not in my vector. Does anyone have any suggestions how I can get a clone successfully ?

I directly cloned cry1A gene p234 expression vector and transformed E.coli Bl21 strain. but expression protein cannot appear.

I am planning to study the overexpressed pattern of lncRNA. Could you explain whether the primer designing procedure for cloning a long non-coding RNA (lncRNA) gene follows the same approach as for protein-coding genes, or are different strategies employed specifically to study the overexpression patterns of lncRNAs?

I have been tring to clone a 25 bp(5`-CACCGXXXX....-3` and 5`-AAACXXXX...C-3`) sgRNA into Px458.

I have been using this method : chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://www.csr-mgh.org/wp-content/uploads/2017/04/CRISPR_Cas9_PX458_protocol-1.pdf

However, I am not using plasmid safe

but so far all the colonies that I sequenced (with U6) have no sgRNA in them. I tried to picked 3,6, and 10 colonies but all of which where empty. has anyone faced such a problem? what should be done? any recommmendations?

Hi, I'm trying to develop a KO cell line from an established cancer cell line. My gene of interest is present in 3 copies in this cell line.

I'm using a multi-sgRNAs technique to increase my chances of a significant deletion. I isolated 4 clones of interest which share a similar trait: they all show 3 different bands after PCR amplification an electrophoresis on agarose gel. This is not so much a concern, since it was one of the expected outcome (the CRISPR/Cas9 system can create three different cutting pattern, resulting in 3 different bands). FYI, the 3 bands are all different in size from the WT band (with the top band being around 100 bp bigger than the bottom lane).

I ran again the sample on a more concentrated agarose gel (2%) with a lower voltage to get nice bands and being able to cut them. I extracted DNA from each band and re-run a PCR on each of them to increase my DNA material. For all my 4 clones, the bottom band amplify to a nice and single band corresponding in size. However, the middle and top lane display the 3 bands again, and it doesn't make sense to me. Indeed, I could understand finding the middle band in the top band sample, or the other way around. But I would have never expected finding the bottom band in the top band sample, because the top and bottom band are clearly separated and shouldn't contaminate each other.

I made a mistake by not using sterile instruments to excise my bands, which could explain in part some contamination. However, if it was this issue, I should have multiple bands in the bottoms sample, which I don't have, and I should have cross-contamination through all the sample, which is not the case. I'm pretty lost so if someone has any idea, I would take the advice with gratitude.

(I attached the gel picture from where I extracted the bands (small gel) and the re-run PCR gel whit the unexplained bands. On the gel, T= Top band; M= Middle band; B= Bottom band).

Thank you all!!

I am trying to clone a large (11kb) and toxic gene in an expression vector. Therefore, I am using the EPI400 CopyCutter strain that is maintaining the plasmid at a low copy number to allow bacteria growth. Attached is the protocol for this strain.

I have to grow the bacteria on plate at 28 C for 2 to 3 days to be able to get colonies.

I did a colony PCR and got 20 positive colonies.

When I put them in liquid culture (5mL to 10mL), some colonies never grown, even at 28 C. Some of them did grow (2 days incubation 28C, 250rpm in aeration culture tube, OD600 above 2.0), but then I only get a really low yield after induction of the high copy production in 100mL culture in flasks.

I tried this several times but still got a really low yield (10ng/uL, maximum 15).

I used this strain for other toxic genes and was able to get more than 40ng/uL (which is around what I need to be able to sequence my plasmid and verify the correct insertion of my gene).

I am using this kit for plasmid extraction Wizard® Plus SV Minipreps DNA Purification Systems

Any suggestions on how I could improve my plasmid yield?

Any suggestions to be able to grow the bacteria in liquid culture after getting them selected on the plate?

Thank you.

I am trying to stitch in a 38 amino acid tag to the N-terminal end of my protein (3200bp) to be cloned into a lentiviral vector (~7000bp). The forward primer for the same, along with the overhang and the restriction site, comes about 150bp long. The first round of amplication gives me a band close to about 3000-3500bp along with a lot of other non specific bands at the higher molecular weight range. I then gel elute this specific band and reamplify using it as a template with the same primers but i end up getting a smear on the gel. I have also tried using this gel eluted sample to proceed with the digestion and ligation with my vector but in vain.

My PCR parameters are as follows:

1. 98 degC- 2min

2. 98 degC- 10s

3. 65 degC- 30s (2-4: x25 cycles)

4. 72 degC- 2min

5. 72 degC- 5min

6. 4 degC- hold

I use Q5 polymerase (strangely, I do not get any amplification with Phusion). I have tried a gradient PCR and it generally works in the range of (58-68 degC). I use about 50ng of the plasmid template for amplification. I understand that really long primers hamper the quality of amplification but unfortunately, this is a necessity right now.

I would really appreciate if anyone with experience can help me out here. My molecular biology is not THAT strong so please point out if I am committing any obvious mistakes.

Thanks in advance!

In 84232 add gene plasmid, I am not sure at which site should I clone guide RNA

In addgene 84323, plasmid they have empty gRNA casette. I am not sure where to clin guide RNA of interest.

1. Is it should be between U6 promoter and gRNA casette or

2. In gRNA casette itself?

Please insight me on Infusion cloning technology and it’s primer designing.

as viruses are notorious for being prone to mutation at high rates its very difficult to clone them though general cloning procedures and cells so in order to solve this problem need specific cell lines

I am trying to do a golden gate cloning but the ligation-digestion seems to be failing

Hello everyone,

I've been trying to clone a bacterial protein from S. aureus into E. coli with pQE30 vector.

After ligation and transformation (with Xl1Blue), I screened my colonies (more than 30) and ended up with just two positive clones. However, one of these clones seems to have both the empty vector and the ligated product (see picture).

Is it possible that this clone during transformatiom did uptake both a re-ligated vector and the vector+insert? I'm pretty sure that I did not pick up two colonies instead of just one.

If they have both of them, can I continue with sequencing to check if my insert is not mutated? If I send to sequence a mixed sample with both the empty vector and vector+insert will it work? And if my insert turns out to be okay and I go on with transformation of BL21 cells and purification will I be able to obtain my protein even if I have an empty vector?

Anyway, I'll try to do another ligation and transformation to obtain more positive clones hopefully.

Thank you in advance!

Homo sapiens clone 55-R precursor miRNA 331, partial sequence

GenBank: PP092201.1

FASTA Graphics

Go to:

LOCUS PP092201 722 bp ds-RNA linear PRI 25-MAR-2024

DEFINITION Homo sapiens clone 55-R precursor miRNA 331, partial sequence.

ACCESSION PP092201

VERSION PP092201.1

KEYWORDS .

SOURCE Homo sapiens (human)

ORGANISM Homo sapiens

Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;

Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini;

Catarrhini; Hominidae; Homo.

REFERENCE 1 (bases 1 to 722)

AUTHORS Al-Rikabi,R.H., Fares,N.H., AbdelWahab,A.A. and Al-Faham,M.A.

TITLE Iraqi patients women with breast cancer

JOURNAL Unpublished

REFERENCE 2 (bases 1 to 722)

AUTHORS Al-Rikabi,R.H., Fares,N.H., AbdelWahab,A.A. and Al-Faham,M.A.

TITLE Direct Submission

JOURNAL Submitted (01-JAN-2024) Biology Dept., Baghdad University, Baghdad,

Baghdad 10011, Iraq

COMMENT ##Assembly-Data-START##

Sequencing Technology :: Sanger dideoxy sequencing

##Assembly-Data-END##

FEATURES Location/Qualifiers

source 1..722

/organism="Homo sapiens"

/mol_type="transcribed RNA"

/isolation_source="patient with breast cancer"

/host="Homo sapiens; sex: female"

/db_xref="taxon:9606"

/clone="55-R"

/country="Iraq"

/collected_by="Rasha Habeeb Fadhil"

precursor_RNA <1..>722

/product="precursor miRNA 331"

ORIGIN

1 taattctatg agatatggct atggatatat atatttcctc tcctatttat atcctaagat

61 tttcttaaag gataatttta ctgttttagg ctttatgttt ttcagtttta tatcatttcc

121 taaagcttag ctacctacaa atgaatatta ttgaagaatt tttctaaaac taaagcatgt

181 cttttataag tttactcttt tataatatcc taaacaaagc atatcctttt ccccttcctt

241 aagtaaattc ttaaggaaat taacttcatt tctaaatcaa agtattttct gcacttttat

301 tgcaccaatc tgttaccttc tgttcatacc atgacttaaa taatgcttac tcaggcacag

361 tactatcagc tgacaacgta cagaaggctc cagaaatgtc actcccatcc cttaaaggtt

421 ggtatatggt cctggtacag tcgtggaggt ccatatgggt gaaaagccca gaagtctacc

481 tgagctgaaa gcactcccaa ggagtttggt tttgtttggg tttgttctag gtatggtccc

541 agggatccca gatcaaacca ggcccctggg cctatccgag aaccaaccta agctcgcgca

601 tcattcctgg aacatcaaga gtgtgaagac tgaagataac ctgaggccta ccagacatcc

661 tatattcacc aagattcaca ttttttggta tgtgtgccta cattttagca aaaattggtt

721 gg

//

I have two different construct:

1. Insert 1 (~3kb) at XhoI and ApaI site in NCVB vector (9.7 kb).

2. Insert 2 (~1.7kb) at XhoI and ApaI site in in pENTR/D vector (2.6kb)

I want to replace Insert 1 with Insert 2. I have already tried restriction digestion followed by Quick ligase (NEB) or T4 ligase (Thermo). I even tried using CIP as I got a large number of self-colonies without it. However, upon CIP usage, no colonies were seen in either plates (self and test). Kindly suggest ways to go about this cloning. The final product that I want is

PS: I am using ultra-competent DH5a (CSHL protocol) for all my cloning.

Hello

I am new to CRISPR-Cas9 and, for my project, I started to collaborate with another lab that claims expertise on this technique. The objective is to produce 3 cell lines, each with a knockout for a different gene. They did all the process of cloning the plasmid until viral transfection, where a point mutation was induced and an antibiotic resistence gene was added and then selected for.

From what I understand, all cells they gave me are resistant to antibiotic but not all cells necessarily have the mutation or the same mutation, and the solution for that, as far as I know, would be single cloning. However, they said single cloning would not be necessary and that all I had to do was run a western blot for the targets in this heterogeneous cell pool to be sure the knockouts worked. They said if the band was weaker or had a different size it would prove it worked. They also said I could already run phenotypic experiments and it would show it worked as well, but, according to them, the definitive proof is the western blot.

Because I am completely new to this, I just wanted to know if such decisions make sense. I just find it a bit weird because I would have to do single cloning anyway in the end. It wouldnt make sense to me to ever try to check the phenotype of a heterogenous cell pool, if I don't even know the mutation rate of the cells, and I assume such rates might even change every time I plate those cells. Moreover, I think that there might be the possibility the protein could maintain the same size even if it became dysfunctional with the knockout.

I found those decisions weird, but I don't have enough experience with the technique to have a solid opinion. Any thoughts?

Previously I have cloned estA gene in pET28b with restriction sites EcoRI and BamHI and stored it in -80˚C. I also proceeded with site directed mutagenesis (SDM) after confirmation of the cloning via PCR gene amplification. However, I didn't get positive result post SDM. Now, as I'm trying to repeat the experiment, I'm unable to exact the plasmid from the previously cloned bacteria and stored as glycerol stock. The bacteria shows antibiotic resistance but when I try to extract plasmid and confirm through gel electrophoresis, I'm not getting any band. Please can anyone guide me and help me troubleshoot the problem.

Greetings esteemed researchers!

I am relatively new to the field and would greatly appreciate some guidance on a challenge I'm encountering in my research.

We're currently engaged in a process akin to cloning, involving Hifi assembly to create linear DNA. The workflow entails N core extension, N core amplification, gene amplification and purification, C frag amplification and purification, Hifi assembly, and final expression and purification.

While following a protocol initially designed for cloning, we've observed that the expression of one particular gene isn't meeting our expectations. Upon consultation, it was suggested that we optimize the Hifi assembly step. (https://www.neb.com/en/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol)

However, given the unique nature of our experiment, I'm unsure where to begin in terms of adjusting the ratios.

Our fragments have varying concentrations, and the molar ratio used for assembly (insert:vector = 2:1) might not be optimal for our setup. Could anyone provide insights or advice on how to approach optimizing these ratios effectively?

Your expertise and assistance in navigating this challenge would be immensely valuable to our research efforts. Thank you in advance for your support and guidance.

Ps. As I'm relatively new to this field, I find it challenging to discern the fragments clearly or to grasp the insights that come with experience. any help is appreciated!

I am planning to do Insitu-hybridisation. I have done the cloning finally . I cloned the PCR product in pGEMT easy vector . after doing the miniprep. I did the restriction digestion with sspI.

size of insert is 500 bp,

pGEMT easy vector- 3015

avaII cuts in insert at 11 positions and in vector at 1533,1755.

after running the gel I got the gel bands at 2000,1256,222 . and 1694,1562,222

which ORIENTATION I should consider for probe preparation.? how to do the linearisation? and How to select the T7 or SP6 polymerase for invitro transcription

Thanks

How about we clone Aaron Bushnell?

I cloned 3HR and 5HR of p.falciparum on a plasmid and the next step is gRNA cloning. I did colony PCR for verification it is perfect but the thing is after taking the toothpeak of the colony in the petri dish should I also stream the leftover bacteria on the tip to a new plate of LB or not? is it necessary? cause the main colony already streamed once and for the second colony PCR do I need to streak it again? I will be thankful if you kindly share your experience.

I am trying to clone 2 genes from bacillus subtilis within a triple strong promoter ( amyQ, amyL,amyE) in E.coli ( both EPI400 and JM109 )with PUC57 and pgD1662 vectors, but I failed for several attempts. as no colony which including my construction survive. I want to ask if the triple promoter is leaky in E.coli and is there a problem with triple strong promoter? as its constitutive? (like changing the E.coli metabolism because of my genes expression?) or the construction ,includes triple promoter and 2 bacillus genes, is not stable in E.coli?

or do I need to change my E.coli strain? do you have any idea of how can I clone my full construction in E.coli? I did not find the problem yet. does anyone has the same experience in this area?

regards

I have a conceptual question. It is a commonly observed phenomenon when using the CRISPR/Cas9 system that edited cells harbor monoallelic knockout. However, in cells stably expressing Cas9 and sgRNA, shouldn't the wildtype allele be eventually targeted and edited?

1. My strain doesn't have a recA knock out.

2. I use a 2 plasmid system for my work. One replicates through rolling circle and the other through theta replication.

2. I have toehold switch-trigger pair on the plasmids which make strong secondary structures and are probably prone to recombination.

3. I get negligible titres for my product of interest.

Is it possible to prevent this problem through simple bioprocess methods (without cloning anything extra)?

I want to clone a gene fragment of around 480 bp in the p-RSET-A vector. To do this, I designed two cloning primers that contain the Bamh I and Hind III restriction sites. The primers worked perfectly in the PCR reaction. Subsequently, I performed a double digestion with the enzymes Bamh I and Hind III and carried out the extraction of the band from a low melting point agarose gel. The vector received the same treatment. I performed the ligation reaction at a 1:3 molar ratio and still got the same number of colonies on the V+ control plate as on the V+insert plate. When I performed the restriction and PCR analysis on the colonies on the v+insert plate, none of them were positive. I clarify that in the design of the cloning primers the addition of 6 bp was taken into account to weaken the restriction sites. Can someone help me?

I am a graduate student doing sub cloning of two similar genes into pBC SK(+); one of the gene was inserted but few colonies appeared on the agar after transformation, the other is not inserting into the plasmid no matter how I elevated the ration of insert to plasmid.

What is the explanation?

Hi, I want to insert 130 bp DNA into 3000 bp vector, however I could not obtain colonies. I used 50 ng insert DNA and also tried to increase insert amount but still I could not observe any colonies. Is there anyone who tried to insert small DNA (nearly 100 bp) and experienced this problem? If so, can you explain your solution?

Hi,

I have been trying to clone 8.4kb miniF replicon from bacmid. I have tried doing that in pFastBAc backbone but the fragment seems to be lost after every trial.

While doing colony PCR, I am able to get amplification from some part of the fragment (1kB or more) but the whole fragment is missing.

I have tried changing ligation ratios, incubation temperature (30 degrees and 37 both). But the miniF replicon is not coming as a whole.

I have also tried to ligate this fragment with 3 other fragments using In-fusion assembly, but the same was observed in those plasmids.

Does anyone know possible reasons of this?

I am currently doing my PhD project which consists of a lot of cloning of new plasmids I am assembling. Our laboratory generally maintains the collection on JM109 strain. But since I am doing a lot of Gibson Assemblies, I have been using electrocompetent DH10B cells for higher efficiency. My question is, can I use standard protocol of preparation of electrocompetent E. coli on JM109 instead of DH10B?

I attempted to clone a small DNA insert (~100 bp after digestion) into a plasmid of unknown size, which originally carried an insert of 111 bp that I removed by double digesting the plasmid. After digesting the plasmid and the insert I wanted to incorporate into the plasmid, I ran an agarose gel to identify and purify the linearized plasmid (see picture on the left). I did not run the gel for the insert. Subsequently, I ligated the digested insert and vector using four different molar ratios of vector to insert (1:1, 1:3, 1:5, and 1:10), using 60 ng of the vector.

Analyzing the obtained results (see picture on the right), I believe that the bands indicated by the green (horizontal) arrow, present only in the lanes of the 1:1 and 1:3 ratios, likely correspond to the linearized plasmid since they exhibit slightly higher molecular weight compared to the bands in the 1:5 and 1:10 ratios. Is that right?

Questions:

1) What do the bands indicated by the orange (diagonal) arrow represent?

2) Why does the incorporation of the insert into the plasmid not increase proportionally with the quantity (in ng) of the insert? I would expect that more vector+insert complexes would form at 1:5 and 1:10 ratios.

2) What could the additional bands indicated by the question mark represent, which are quite intense in the 1:1 and 3:1 ratios but absent in the other two ratios?

My IPSC clones (not estbished lines) are fargile that when i transfect them with Cas9 palsmids - the cells die. I have used lipofectamine, and electroporation method for transfection. But cells dont survive.

what should i do different?

I ran restriction digestion on my plasmid just to verify my inserts and send for sequencing. my backbone plasmid is PL6 with 9843 bp containing a 3HR (877bp) and 5HR (782). My enzymes for 3HR is EcorI and NcoI and for 5HR is SacII and AFIII. I digest my inserts in different separate tubes. For 3HR for example I get two bands if check for the size the size of my insert is correct but there is another band a bit lower than plasmid size which is almost 3kb. so the first band which is plasmid is almost 8kb the second one is almost 3kb and the last one is around 800bp (my insert size) . Do you have any experience that where the problem is? since I get three bands instead of two bands (my plasmid and insert only) I cannot verify it. I appreciate your answers

I'm attempting to use Gateway technology developed by Invitrogen to introduce my gene of interest into a PLenti vector. The procedure involves shuttling the gene of interest from entry vector to the destination vector using their specifically designed sites (namely attL1-attL2 for entry vector and attR1-attR2 for destination vector) and an enzyme provided in the kit. I'm advised that this technology is really popular because once you successfully clone the gene of interest into entry vector, you can virtually shuttle this gene to any destination vector that contains attR1 and attR2 sites using a 1 hour reaction. I have had some trouble cloning the gene into the entry vector, and I am wondering whether I can just clone it directly into the destination vector.

Has anyone experienced this method before?

Hi,

Thank you in advance.

I have a glycerol stock of a mutated E. coli (point mutation). I am wondering, if I streak a fresh plate (LB ampicillin) will I have the same clone as original. So that I don't need to send the miniprep plasmid DNA for sequencing?

Hey there, currently I am working on threonine synthase enzyme of Leishmania. I have successfully cloned it into E.coli DH5alpha strain. I have also transformed it into E.coli BL21(DE3) strain. I tried induction at 37°C and 25°C with IPTG concentration 0.1mM, 0.5mM and 1mM respectively. At 25°C with IPTG concentration 0.1mM after purification I got very faint band of expected size ~75kDa and it was confirmed by western blot analysis too. But the problem is that yield is very low. How I can maximize the yield? How the recombinant protein expression is increased in E.coli BL21(DE3) strain? Can anyone suggest paper on this ?

Some context:

I'm trying to generate a clone possessing a gram positive phage endolysin gene in E. coli DH5alpha.

I'm using the pet28 vector and initially tried to clone/transform directly into both JM109(DE3) and pLysS with no success.

I can confirm the ligations are working with amplification of the cloning site.

I have even tried fusing the gene to GFP, constructing the pet28_GFP vector first and once I try adding this endolysin I fail to get clones.

Can a gene lead to complete plasmid instability in cloning strains that doesn't possess T7 RNAP which T7 vector systems rely on for expression? I am aware of T7 lysozyme repressing T7 RNAP and maybe there is a possible of this gene performing a similar role

I'm aware the protein is toxic but surely one should be able to generate a library to see it all go wrong in expression attempts.

Could my problems lie in DNA secondary structures effecting plasmid stability (replication)? any insights would be great.

Hello everyone,

I have a recombinant clone (My gene fragment, 2 restriction enzyme sites and pet 28a vector) that gets synthesized by a company. Then I added 20ul DEPC water to that 5ug clone and stored it in a -20 deep fridge. After that, I ordered a Thermo Scientific Competent BL21 cells kit. Afterwards, I added 3ul of my clone into 50ul of competent cells, incubated it in ice for 30 mins, heat shock it for 30 sec, then again incubated it for 2 mins, and put the cells tube in a shaking incubator for 1 hour. After 1 hour spread the cells (containing clone) on LB+agar plate containing ampicillin. The next day, a few cells grew in that plate and for the confirmation of the transformant I performed Colony PCR, but the colony PCR results were not good and bands didn't appear.

Kindly let me know what to do next or how to confirm the transformant.

Hello,

I tried to clone 3 insert into plasmid pUC18. I used Takara infusion cloning with this reaction:

1 μl 5X In-Fusion HD Enzyme Premix

0.59 μl Linearized vector (2667bp, DNA mass=44.35 ng, conc=179.1 ng/ul)

0.85 μl Purified PCR fragment I (3740 bp, DNA mass=62.19 ng, conc=175.4 ng/ul)

0.77 μl Purified PCR fragment II (482 bp, DNA mass=16.03 ng, conc=99.2 ng/ul)

1.84 μl Purified PCR fragment III (1694 bp, DNA mass=28.17 ng, conc=36.6 ng/ul)

In total I have 5ul reaction volume and do the infusion reaction for 20 minutes in 50 degree C.

I used NEB calculator to get my DNA mass. Is my calculation correct. I search in takara website they have their own calculator to count the DNA mass. Please give me suggestion is there something wrong with my method? I have been failed working with this clone for 3 months.

Thanks

Hi,

I'm facing a recurent problem with the cloning of a given cDNA. Everytime I'm doing the cloning I end up with a mix of big and small colonies on the insert+vector plate while getting only few big ones on the vector alone plate. The small ones are clearly not satellites, I know how do they look. I picked some of the small one but they are generally not growing in liquid. Tried to grow plates at 30°C no changes. Ran my ligation on gels and they look very efficient. I clearly don't know what to do. Any suggestions? Thanks

I found out that the water I used to dissolve my DNA after phenol-chloroform extraction was contaminated with bacteria (not sure which, but likely E.coli used for cloning). Can I remove these bacteria by simply performing another round of phenol-chloroform extraction on this sample? Thanks!

Also, does anyone have an idea how big your insert can be in a 6 kb vector when you use the gibson assembly as the cloning method to create a midigen and where you only design a forward primer at the beginning and a reverse primer at the end of your insert?

I have now developed a midigen with an insert of 4 kb (NF1 gene exon 20 to 24 with introns) in a pSPL3 vector of 6 kb, so together that is 10 kb. I did this using the Gibson Assembly cloning method. I designed a forward primer in intron 19 and the reverse primer in intron 24. For sequencing, I designed new primers to divide the 4 kb piece into pieces.

I am trying to develop midigene to study larger splicing effects with the mini/midigen exon-trap assay.

I have been trying to express my construct for the last couple of weeks but after several attempts, it's still failing although similar parts (five inserts for golden gate assembly) have been used by my colleague in past and it worked. I want to know if I am doing something wrong with my ligation reactions or digestion of parts is working fine despite I have checked protocols several times. Here, I would like to request the expertise if anyone can suggest options which I can use to make sure I following the right path.

Thank you in advance.

Hi, I just have been in fusion cloning.

I want to insert my GOI in pMIG II vector.

So, I amplified my GOI using PCR with 15bp overhang (PFU used), and linearized the pMIG II vector with EcoRI and XhoI.

(PCR fragment and linearized vector were cleaned up using gel elusion kit)

I used Stbl3 for transformation.

And I used in fusion molar ratio calculator to determine the DNA concentration of fragment (1728bp) and vector (6512bp).

But, I failed to get plasmid with my GOI. and I sequenced this failed plasmid and found that this plasmid matched the sequence of the intact pMIG II.

(E.coli did not grow in linearized vector only control)

How do I solve this problem?

Hi everyone!

I'm struggling to find the correct English translation for "surclones".

For example, you can obtain these "surclones" by streaking an [ADE-] strain on an adenin-depleted media : the majority won't grow but you can see a few clones appear due for exemple to the reversion of a mutation. So how do you call these few clones ?

Thank you all!

I would like to ask a question about constructing stable cell lines.

If someone has the whole genome sequencing results of their overexpression stable cell line that would be really helpful. That would give us a clear solid example of what going on during the fregment integration steps.

Thank you for your reading, and please let me know if I did not make my idea clear. It would be really helpful if you answered my question.

Best,

Le

I require the empty vector of this plasmid: https://www.addgene.org/40346/

The ends have been destroyed:

How do you suggest to do this?

Hello everyone,

Is there anyone who has cloned with pAcGP67A secretion vector with GP67A signal peptide.

I tried to clone with insert 1.2kb in the vector, MCS: BamHI and NotI. C-term Thrombin and His-tagged was engineered.

I keep getting a stop codon after the signal sequence before the BamHI then my insert at the N-terminal.

I simulate with Snapgene but i keep getting inframe with GP67A and a stop codon appears .

Please i need a help.

Thank you

Hi, I have recently started my cloning experments and don't have much experience in doing ligation reactions. I have five insert parts with each having 10ng/ul concentration and i need 200ng of inserts in my final 20ul PCR reactions with 50ng of plasmid. But with this requirement the need of inserts itself will be around 20ul which is not making sense to me.

Therefore, to achieve these reactions, what adjustments do adjustments toadjustmentstoI have to consider for proper calculations, do i need make any adjustment with total volume of reaction or something else. Please guide me with this some explanation if possible as i am a bit confused with this situation.

Thank you in advance.

Someone who can give advice on this? I wanna use a midigene for a exon-trap assay. I read in several articles (such as: Sangermano et al., 2017 https://doi.org/10.1101/gr.226621.117) that the gateway cloning methode is used for creating a midigene, but wouldn't the gibson methode also be possible and why would you choose one and not the other?

Unwanted base pair insertions/detetions!

This is an age old story with this cloning journey for one particular plasmid ~15kB with a CMV promotor. I get unique base single base pair deletions/insertions that I am pretty sure are happening during bacterial replication, not as a result of a bad ligation, etc.

Details:

-E.Coli DH5a competant cells and heat shock transformation.

-In several stages of cloning, of 6 screened bacterial colonies, all differ by base insertion/deletion, different location in every clone, breaking my reading frame and rendering the construct useless.

Most recently, I screen a clone (12) and confirmed the sequence of my cDNA is 100% correct and there is just 1 moleular species present.

I want more material, so I go to my agar plate with the streaked bacterial colony of clone 12 use this to innoculate my LB + antibiotic. I grow for 2 days at 30' as it the plasmid is large (15 kB) and I worry a bit toxic to the cells.

After miniprepping this culture, sequencing reveals a new base deletion in my protein that didn't exist in the original parent cDNA.

-Any tips? I have tried growing at 37' for 16 hours, 2 days for 30', leaving it a room temp for a day to start and THEN putting in 30', but I'm not sure what else to try and need to produce more of this construct one way or another.

Dear colleaques

I have a problem with cloning of my PCR product (829 bp) into pET101 TOPO vector (5753 bp) by Invitrogen™ Champion™ pET101 Directional TOPO™ Expression Kit. I performed a PCR reaction to obtain my product, which I purified from the gel. The forward primer contains the CACC sequence as recommended. For PCR reaction I use Phusion Plus DNA polymerase whitch generates blunt ends. I got strong band with corect size and purified my DNA by Nucleospin kit. The concentration of my PCR product was 35,2 ng/ul.

For cloning reaction I follow the instructions in manual. It is important to use 0,5:1 to 2:1 molar ratio of PCR product:TOPO vector. So if I used 1 ul of vector (15-20 ng) I dilute my PCR product to concentration 3,52 ng/ul and add 0,82 ul of them into the cloning reaction (molar ratio 1:1) and incubated for 5 min (first time and 20 min second time with the same results). As control I used reaction only with vector (without PCR product). With reactions I transform One Shot TOP10 Chemically Competent E. coli and incubated on the agar plates with ampicilin (100ug/ml) owernight at 37°C. But the results were the same on both plates, where I got hundreds of colonies.

Then I took some 20 colonies and used them as templat in control PCR wth the same primers which I used at the beginning of the process. Reasults were negativ. This was also confirmed by restriction analysis after DNA isolation (miniprep).

Where could be mistake? Thank you very much for any advice.

Hello,

I am transfecting linear DNA along with an Adenovirus transduction to HEK cells and need to isolate the DNA from both the linearised plasmid and viral genome at different passages for restriction digest analysis. I am not interested in the nuclear DNA

Total DNA extraction will include all genomic DNA which I fear will interfere with the restriction digest and produce a highly visible smear on the agarose gel. Ideally, I would want to just isolate cytoplasmic DNA.

Will it be okay to use the total DNA? Could I isolate extrachromosomal DNA using a standard miniprep kit, although they are meant for bacteria? Or would it be better to perform cytosolic isolation followed by DNA analysis?

I am doing cloning of a big bacterial insert (3705bp) into a vectors of varying sizes ranging from 3017bp to 3469bp for my bacterial two hybrid experiment. Among other problems with my cloning I have noticed that neither 1% nor 1.2% agarose gel effectively separates my 1kb gene ruler ladder. So, I went for 0.7% agarose gel but I am facing an issue with the time required to run this gel. It runs so slower for me. It took me about 2 hrs (10Volts) to run it so that it crosses the center and reaches close to the bottom part of the gel.

I wonder that less agarose should make the gel run faster but why is it the opposite for me. Can anyone advice or give suggestions on potential factors to investigate or methods to improve migration speed or it is normal to take longer time.

If anyone has expertise in cloning big inserts in the bacterial two hybrid plasmids I would greatly appreciate some tips and suggestions to be successful in cloning.

Thank you in advance for your time and help.

How will Matthew Perry Pass on His Genes? Maybe he cannot now because he died. But we may be able to clone him or take his sperm.

Hello everyone,

I'm currently facing a challenge in my cloning strategy involving the ccdB gene. Despite ordering a gene block with a J13119 and a standard RBS, I've encountered a lack of success after three rounds of cloning using ThermoFisher's ccdB survival strain.

My working hypothesis is that the toxin is being overexpressed, leading to the inability of bacterial cells to metabolize it effectively. To overcome this issue, I'm seeking alternative promoters or sequences that can provide a smaller, more versatile solution than the current chloramphenicol-resistance + ccdb gene from Thermo. Ideally, my sequence should be compatible with both gateway cloning and standard restriction cloning techniques.

Any insights or suggestions regarding suitable promoters or sequences for my specific case would be greatly appreciated.

Thank you for your time,

Juliano

I use restriction enzyme cloning method, and I have been using new reagents e.g., competent cells, and ligase reagent because initially I thought the problem is that these reagents were outdated. Now with these reagents I attempted to clone my shRNA into the vector with 1:3 DNA:insert ratio, but I didn't even see a single colony.

from where the transcription of the gene in the vector starts , is it always from 5 to 3 or it depends on the direction of the promoter .

Good day everyone, Please how do I select a single clone, from a population of positive cells, I have done puromycin selection and now I want just a single clone from the cell population but I am having difficulties in doing this, please how can this be done. I will appreciate your kind response please. Thanks in anticipation !

I tried and cloned the gene in the 3xFLAG-APEX2-NES vector. i am transfecting 1.5ug of each plasmid in HEK293T cells in 12 well plates at 80% confluency then after 48 hrs of transfection I prepared lysate using RIPA+PIC. on probing with flag antibody only empty vector control is expressed not the fused protein. I have confirmed the clone by sequencing also.

Please help me out with the problem of why fusion protein is not expressed.

thank you

I extracted DNA from several transformants, but they showed really degraded smear bands. Here're the steps of transformation:

I used Infusion Snap Assembly to ligate a 16kb linearized plasmid fragment, a 100bp fragment, and a 300bp fragment. The linearized backbone undergoes gel extraction and has blunt end. The inserts are overhang-added by PCR, and I also took them from gel extraction.

Then I did bacterial transformation using NEB dh5alpha high efficiency competent cells. Few colonies grew on 50ug/ml spectinomycin plate, 30C in 40hrs. They were picked and inoculated in 5mL LB broth, 50ug/ml spectinomycin, 37C in 22hrs 180rpm shaking. Then I did QIAGEN Miniprep for them.

I used 50ul water for the dilutions, and nanodrop reading shows ~1.8 260/280, and ~2 260/230. However, when I checked the undigested plasmid DNA on the gel, they showed really degraded band. I used the same kit for my 19kB backbone plasmid, and it worked well. When I did digest them, the band looked worse. Also, it's weird that the undigested DNA showed two bands, a large band that degrades a lot, and another small ~500bp linear band.

Therefore, I'm asking for suggestions to improve the result. Could it be the problem of the ligation, transformation, or miniprep? I attached the gel image of the undigested DNA.

how to add two restriction enzyme sites to primers for cloning a gene sequence (size 1-2kb) to pGEM-T Easy Vector followed by pcambia 1301 and is it necessary to add setting sequence before the restriction sites ?

Hi,

1. Does any one know what is the maximum length of amplicons we can amplify using PCR? I need to amplify fragments for assembling a big vector (approximately 100kb).

2. Do you think it's achievable if I amplify 10 genes of approximately 10kB and stitch them together using GIBSON assembly?

Any recommendations/suggestions are appreciated.

Thanks

I have cloned a gene with EcoRI/BamHI restriction sites into p3XFLAG-CMV-14. The sequencing has confirmed that the gene has been inserted in the right direction. For some reason, I would like to reverse the direction of the gene in the same vector backbone. Is there any method to do it?

Thanks in advance!

Hello, dear researchers. I am interested in a gene in Pombe. The gene contains 2 introns. I want to overexpress this gene in Pombe. Can I amplify and clone the gene by PCR or do I need to do cDNA translation first? Since the gene already belongs to pombe, will the presence of introns cause any problems?