Cloning - Science topic

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 expired in our lab. 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 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.

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

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?

Hi all,

I'm attempting to clone a GC rich insert (500bp) into a vector that is approximately 5kb and also GC rich. After sequential digests (one enzyme works at 37 while the other works at 65 degrees Celsius), a 0.5% agarose gel reveals that the vector was efficiently cut as indicated by a 500bp shift down of the parent insert vector. Oddly, the 500 bp insert is barely visible. When blown out, the gel shows a smear near the 500 bp region. Is there a reason this is occurring? We are struggling to get any colonies to appear for diagnostic digests so any help would be appreciated.

Thank you!

I have an insert size of 900 bp, trying to clone it in pET28a vector. But after every the transformation when i am doing colony pcr and digestion check i can only get the fragment of 500 bp. Why is it so? The restriction sites are BamHI and SacI and the gene doesn't have internal sites of these, which i could know by sequencing. So why is it so. Do i need to subclone instead of directly trying to clone in pET28a plasmid?

Hello everybody,

I'm having trouble cloning a 700bp insert into a 18kb plasmid. I'm using Neb Hifi assembly (Gibson assembly) but everytime I have no colonies on the plate. I tried many different insert/plasmid ratio but nothing change. I'm using XL10-Gold bacteria. Both insert and plasmid were purified on agarose gel.

Thank you so much.

For the Gibson cloning into pH-ePPE vector (19kb), I use NEB Hifi builder mix with 400ng of vector backbone (18kb) and 10ng of 250bp insert and NEB chemically competent 10beta cells for transformation. I know my Gibson assembly is working as I have confirmed by PCR. I have used 1ul to 10 ul of Gibson product as well as 1ul of 1:3 diluted product, but I am not getting a single colony post transformation.

Can anybody suggest how to troubleshoot this problem.

Hello

Good time everyone

I ran the cloning product on a 1% gel and observed the following band. Can anyone tell me what is the reason why my band has widened?

I have a insert of 400 bp cloned in vector pbluescript II KS (+) of size 3.0kb at RE sites XbaI and XhoI. But when I try to double digest the plasmid it is not happening. I am sharing picture of result showing the same. Please can anyone provide me the reason and solution for this.

Fig: Lane1: 100 bp plus ladder; lane 2: plasmid double digested; lane 3: plasmid single digested; lane 4: uncut plasmid

I was working on a gene construct synthesized in pet29 vector as a clone. Primers were prepared and optimized with gene at Tm 58 degrees. Once primers were optimized, I carried out transformation in expression vector and checked colony PCR with the same set of primers. After some time, I needed to conduct PCR for the same gene again for TA cloning and repeated BL21 transformation but issues occured. My primers that were previously optimized didn't work on the same gene on the mentioned Tm. After numerous trouble-shootings, I decided to check either the problem has appeared in my gene construct or not. I checked my commercially synthesized cloned gene on agarose gel in the intact and digested form and there was no band of gene once visualized. Is there any chance that my clone is destroyed by nucleases? What can be the reason for such conditions? It will be a great help if you can guide me

I was working on a gene construct synthesized in pet29 vector as a clone. Primers were prepared and optimized with gene at Tm 58 degrees. Once primers were optimized, I carried out transformation in expression vector and checked colony PCR with the same set of primers. After some time, I needed to conduct PCR for the same gene again for TA cloning and repeated BL21 transformation but issues occured. My primers that were previously optimized didn't work on the same gene on the mentioned Tm. After numerous trouble-shootings, I decided to check either the problem has appeared in my gene construct or not. I checked my commercially synthesized cloned gene on agarose gel in the intact and digested form and there was no band of gene once visualized. Is there any chance that my clone is destroyed by nucleases? What can be the reason for such conditions? It will be a great help if you can guide me

Is there any online tool available to check, if designed primers are suitable to get overexpression of the histidine-tagged protein, cloned in pET28a vector?

How to confirm the suitability of primers for the same.

I would like to find out the amino acid sequence for a few CD3 antibodies. Does anyone also know any platform/database that I can find amino acid sequence of antibodies?

Need to add a gfp tag as well.

I am trying to use double selection marker, G418/kanR and BleoR in pPICZalpha plasmid. I am constructing the plasmid with both antibiotic resistance gene and clone it into E. coli Top10. However, I cannot get any colonies in LB low salt KanR BleoR plate. I only know that LB low salt plate is required for BleoR in E. coli. Is there anything else wrong? Any suggestion is welcome and THanks!

I recently ordered a cloning product from Thermo Fischer, and the instructions for primer design require that the forward primer have the Kozak sequence ([G/A]NNATGG) on the 5' end. However, I have never designed primers requiring this sequence. How do I design the primer? I made two examples below, but I am not sure which one is correct:

1. GagatctgtcaagagaatccATGG

2. AAATGagatctgtcaagagaatcc

My aim is to identify and amplify the variable regions of mouse Ig and to further clone the sequences in a suitable expression system.

We are interested in transfecting a CHO cell line to produce a recombinant protein.

We are planning to use the limiting dilution method in 96-well plates to select single-cell clones to be screened for expression and we are looking for a detailed protocol for this cell line. Specifically, we would also be interest to know the cloning efficiency with this cell line (i.e. the expected ratio between wells plated and clones obtained).

 

I want to know about the impacts of cloning process

It is possible to transfer genes from plants to humans. This is done through a process called genetic engineering. Genetic engineering is a powerful technology that can be used to improve human health, but it is important to use it carefully. There are potential risks associated with genetic engineering, such as the possibility of unintended consequences.

How the gene cloning work and mention few steps?

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 want to digest pET28a plasmid (5369 bp) with 2 restriction enzymes (RE)- EcoRI and XhoI for cloning. I checked the REs have single cutting site of each in the plasmid. However when I digest it with EcoRI for 2 hours, it gives 2 bands- 5369 bp (desired) and ~5000 bp (lane 1).

If the construct has an insert that created another EcoRI site then it is now ~10kb plasmid. Interestingly, XhoI digests the plasmid completely (loaded full reaction volume) and gives one sharp band of desired size (5369 bp) not 10kbp (lane 2).

And again in case of double digestion, the undesired band appears again (lane 3).

If it is supercoiled plasmid, then why does it appear in case of EcoRI only? I changed EcoRI brands, incubation time, buffers..but same result.

pET28a: 14ug

EcoRI (invitrogen): 20U

XhoI (NEB):20U

10x Cutsmart buffer (NEB): 2 ul

Rest volume: NF Water

Hi all, is it normal after conduct an E.coli transformation, there were colonies that having low producing protein target and the other having higher producing? What factor(s) that affect this result? Thank you.

I purchased the human clone of this gene and sub cloned it into a Xenopus oocyte plasmid (it contains Xenopus oocyte beta globin to enhance the expression in Xenopus oocyte) with a polyA site. But no functional activity of this protein was detected (I repeated the experiment 3 times). I sequenced the clone and it is correct.

From the literature, I know that its rodent and fish homologues, and some of its family members in human were successfully expressed in Xenopus oocyte with detectable activities without expressing their known co-factor(s). I found an unpublished dissertation work online saying that they couldn't detect function of this human clone in Xenopus oocyte either using a different functional assay.

I ordered antibody of this protein hoping to see whether it didn't express or its function was inhibited for some reason. While waiting for the antibody (it will take weeks), is there anything that I can do to help figure out the reason why I didn't detect its function?

Hi, I try to clone a V5-TurboID (1Kb) into a roughly 6kb big backbone (pEGFPC2). The restriction enzymes used are NheI and XhoI. Both fragments are gel purified and ligated with T4 Ligase (1h or 16°C overnight), then transformed to Dh5alpha. I can not get any colonies. Ligation ratios varied from 1:3 up to 1:10. Does anyone have any ideas how to solve the problem?

I have to clone a cDNA to insert in pcdna3.1. The forward primer generated is having tm 83 and reverse primer has tm 67. I am not getting amplification from normal pcr, gradient pcr, hotstart pcr and also touchdown pcr. What should I do? Is it possible to amplify cdna with primer having this much temperature difference?

We cloned a viral gene into pET28a expression vector and expressed in E. coli. Then, expressed protein was examined by WB and other methods. However, unexpectedly, molecular weight (MW~35KD) of the expressed protein was found to be higher than its predicted (expected~30KD) MW. How could I explain it and what are the major reasons behind it?

Hello,

I previously transfected (by using lipofectamine) rat hepatoma cells with my target gene and used 10%DMEM+PS+500 μg /ml G418 to obtain clones that were expressing my target gene. After a while my negative control cells were dead as expected because they were not transfected and I observed clones in flasks, also as expected. However, I recently started working with HepG2 cells and this method would not work. I checked for transfection using GFP as a positive control under the fluorescent microscope but non-transfected cells (my negative control) would not die, they were just growing fine in G418. I even tried to increase the amount of G418 but this time my transfected cells started to die too. What could be the reason for the non-transfected cells to keep proliferating in G418?

Thank you!

Hello scientific community!

I have been trying to establish an IF staining for CD36 in MECs (cardiac, pulmonary and renal glomerular) using CD36:APC (clone REA670; Miltenyi biotec) and CD36:PE (clone CB38; BD Biosciences). But no success so far, even in blood smear stainings.

I have tried different concentrations of each Ab with incubation 4°C ON and 37°C at RT.

Does anyone has experience with CD36 staining? I mostly find successful FACS protocols in publications, but not cell staining.

Thank you!

I used KAPA hifi pcr kit to linearize the plasmid for in-fusion cloning.

Following the protocol, the DNA template amount was 5 ng and I already diluted it 100x. Annealing temperature: 66 69.

The primer was designed on TAKARA website.

Gel electrophoresis method: 120V 60min

To induce Notch signalling in Huh 7 cells, I plan on cloning the NICD and then using it to activate the Notch Pathway. I need the region in the DNA that specifically codes for the Notch 1 intracellular DNA.

BAC vectors are use for cloning purpose and are good for cloning large fragments though they have low copy numbers.

when I am expressing my cloned gene, i am getting the desired band in clone induced but the vector induced is also having a band on the same position but faint.

I am trying to clone 2461 bp fragment (restriction enzyme sites are included) into pET28a+ plasmid (which has his-tag that i will be using for purification of protein of interest later). So far, amplification of gene and digestion were completed. Then for ligation step, different V:I ratios were tried and these are 1:1 1:2 1:3 1:5 1:7. Not a single colony was obtained after transformation of ligated product into competent E.coli DH5a. This cycle was repeated multiple times.

What should i do after this point? Is the fragment too large for this vector or can i try a different V:I ratio again or should i use a commercial cloning kit? If so, which kits can be used in this case?

P.S: I have recently used these restriction enzymes and competent bacteria for another cloning experiment and there is no sign of contamination or faulty.

Hi everyone

I'm trying to clone a gene sequence. I have designed specific primers on the gene sequence, but the PCR has more and more bands. I tried to clone, and later did a PCR with both sequence-specific primers and M13 universal primers, obtaining more bands on the gel. How do I get my one band? Do I have to perform the PCR? What do you suggest? Has anyone had this problem before?

Hello Everyone,

I am planing to conduct a Yeast One-Hybrid screen using the Takara Matchmaker Y1H Gold screening kit.

Reading trough the manual I did not really find information on how this strategy would ensure, that the cDNA library clones are translated in frame with the fused Gal4 activation domain. There is only one section elaborating on this problem saying, that yeast can tolerate frame shifts and as I understand still expresses the right protein.

As I remember in the past there used to be vector systems, where you could insert your cDNA clones in different frames (e.g. +1, +2, +3) and therefore one of the three vectors resulted an in-frame protein fusion. (for clarity: in such case you had to prepare 3 libraries one with each vector)

Maybe there is a trick during the SMART cDNA synthesis. I mean that the adaptors which are fused to the cDNA library clones for recombination cloning are ensuring that every third of the cloned transcripts are cloned in a different frame.

I would appreciate if anyone can clarify this question for me.

Hello,

I am designing a plasmid with an SV40 promoter-driven antibiotic resistance. Does expression from an SV40 promoter require a TATA box upstream of the transcription start site? The original vector had a TATA box at -30, however this is lost in my cloning strategy. With my current plan, the transcription start site is just 8bp from the end of the SV40 promoter. Will this allow for expression, or is a TATA box needed?

Thanks!

I am trying to clone my gene of interest in plasmid but the colonies appear only on 25ug/ml Amp agar plates and not higher then that. Also when I am trying to grow the colonies in 25ug/ml Amp LB broth, no bacterial growth is observed. What could be the possible reason? And any solutions?

I have two linear DNA fragments that will be ligated together (each ~700 bp).

This fragment then needs to be ligated into a linearised vector (plasmid).

Would I need to perform a clean up on the first reaction, if it will be directly used for a successive ligation reaction?

as i cloned promoter region in topo vector but somehow only 18oobp region was cloned instead of 2000bp

Hi there,

Is there anybody who can possibly share to me the sequence of the following plasmid: pMDC163, I need it in FASTA format please. I've only found the image of the plasmid but I can't find the sequence, I need it for a cloning process. I would really appreciate it if you could help me.

Hi, I need to amplify a tRNA fragment in a normal PCR,

The primers I designed suppose to give me a product of 40 base pairs. I want to transform it into pGEM and send it to Sanger sequencing . My question is what is the minimum size of PCR product that needs to be cloned and then sent to sequencing in order to get accurate sequencing?

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?

I have been trying to clone a coding region of my gene in pdonr using gateway method. I successfully cloned CDS but when I sequenced the colony the region from the middle of about 100 BP was missing. What could be the possibly reason of that? Note: It doesn't have any other transcript. What should I do to overcome the issue?

Similarly, for promoter and coding region together, get colonies on zeocin resistant plates but I don't get positive colony PCR with specific primers. What should I do?

Available products carry mutations in endonuclease genes cause they are used for cloning applications. Please advise. Thank you!

I have to clone a gene below its native promoter but it is part of an operon, so I need to amplify the two genes separately and clone it in two steps.

I found a plasmid in my lab which already has the same gene cloned downstream of the native promoter but after analyzing the sequence, I found that there are two additional bases between the promoter and the START codon apart from the restriction site in between. I can perform PCR and amplify both sequences together by designing primers but the additional bases are going to increase the distance between the promoter and the gene. Would that create major problems during transcription?

Thank you.

i am cloning my gene of interest in vector pet32 and pgex4t. upon transformation i got colony on antibiotic containing LB Agar. also colony pcr and pcr with the plasmid extracted from the clone showed the desired band on agrose gel electrophoresis. but when i was inducing them. i didn't got expression of that particular protein. please explain me reason behind it

I need to clone ftsZ gene from Mycoplasma gallisepticum. But the gene has 2 stop codons inside of it. I was told to use Gibson assembly, but I'm not sure how.

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?

Searching for the correct monoclonal antibody has been quite hard, because, although in most cases companies specify the region/epitope of the antibody (e.g. extracellular domain, 1-20 aa etc), in others there is only the clone id and no more information.

In particular, I am looking for the epitope of TR75-54 or 54.7 clone of a TNFR2 monoclonal Ab. Any advice would be valuable! Thank you!

Since lentiviruses are known to preferentially integrate into the transcriptionally active sites in the genome, I am wondering if there was a simple and effective strategy to identify clones that have the lentiviral insert integrated into the genome but not on any site with functional consequence. I would really appreciate any helpful suggestions.

Thanks

What are the benefits of adding a pre print of Article in RG ? Will the there any issue in publishing online later on ?

Hello All,

I'm facing a problem while transforming genes into P. pastoris. Actually, I'm trying to express an antibody heterologously in P. pastoris. So, for that I engineered the vector pPIZalphaA in such a way that two genes (heavy and light chain gene) of the antibody were inserted under two AOX1 promoters individually. Then I cloned the vector into DH5alpha, confirmed the colonies, and then isolated the plasmid. After linearization of plasmid, I transformed it into P. pastoris GS115 via chemical and electroporation both the methods. After 2-3days, I got several colonies, and I cofirmed the clones by PCR using heavy and light chain specific primers via colony pcr and pcr with genomic DNA of those clones. Mostly, all the colonies were confirmed with light chain integration and barely 2-3 colonies were confirmed with the integration of both light and heavy chain genes. Then, I selected those 2-3 colonies for further expression studies and initially I got expression (secretion into the medium) of the antibody (~150 kDa) which was confirmed by SDS-PAGE. After certain time (approximately 20-25 days) when I performed the same expression studies again, I didn't get the expression from the the same clones. Then I did genomic DNA extraction of the same clones and did PCR with heavy and light chain primers. I got the amplification of light chain gene only, heavy chain amplification was missing. Simillarly, I made fresh recombinants also and again I observed that after certain time, heavy chain gene amplification was missing. So, I'm not getting why the heavy chain gene is missed out, or else it is becoming unstable within the genome of Pichia after certain generations. I need some expert's opinion on this matter.

Thank you

Hello everyone, I have one doubt regarding cloning ?

I am using pET30a (+) vector for cloning (5422bp) and Insert size 804bp. I tried all the ligation reaction ratio lilke 1:3, 1:5, 1;7 (100ng vector and 300ng Insert) but some time I got positive colonies but the problems is that whenever i am going to isolate the plasmid, I donot found the plasmid in the positve colonies

Please give me some suggestion

what should I do for it

I was thinking what if we use the different promoters for any gene which is being cloned in living organisms? For example what if we use the promoter of a different gene for a different gene which is being cloned? what results can be observed? Wither would it overexpress the gene or downregulate it?

while doing klenow reaction for blunt end cloning,i am not getting why it is necessary to add dNTPS to chew 3' overhangs

One of the projects that I am working on calls for the need for cloning and transformation in cotton. For this project, I plan to perform RNAi-mediated gene silencing using pHELLSGATE 4.

I cloned a fairly large cDNA into a pcDps backbone previously modified with a BsrGI restriction site. I was able to cut the cDNA out of a plasmid with the respective enzyme and inserted it into the vector. I performed a test digest with BsrGI and got the expected product.

However, after performing a test digest with BsrGI and BsaI, I noticed that one band was missing. This was always the lowest band, even after I repeated it with a different enzyme that cut into both the backbone and the construct.

Does anyone have any idea what this could cause because I actually assume that the construct is intact when I remove it from another plasmid?

Thanks for help :)

Hello,

I have performed some recombineering protocols and realised that the chances of my plasmid being in a multimeric state are quite high.

I previously designed 7 primer pairs that will produce alternating amplicons of 500 and 700 bp around my recombineered plasmid (which is 35kb) just so that I could get an idea that no weird recombination events occurred when looking at it in a gel.

Anyways, I did the 7 PCR reactions on a control with the original plasmid, and they produced the expected pattern, but when performing it on my miniprep-purified plasmid I was obtaining a lot of bands of all sorts of sizes (larger and shorter than expected amplicon). Funny thing is that these multiple bands seemed to follow the same pattern in all my replicates (different pattern for each primer of course, but same throughout the different colonies tested) which makes me rule out the possibility of salt contaminants affecting primer binding etc. I thought it might be bacterial genomic contamination that was being amplified, so I performed a CsCl-ethidium bromide density gradient to purify it and sent it off for sequencing.

But now Im wondering, would a multimeric plasmid yield multiple bands if amplified with a single pair of primers?

By the way, I can't run it on a gel to assess if it's multimeric because of its large size 35kb, although I am going to ask if anyone at my lab has a pulse field gel electrophoresis just in case.

Thanks!

I had sent my article to a journal, but then I realised it was a fake one or a clone and I withdrew my paper. Now, I wish to delete it. The title is ADVOCATING DEEP ECOLOGY IN MASURKAR'S SHERNI. By mistake, I chose the option IT IS NOT MY ARTICLE. Now I want to permanently delete this article. How can I do so? Kindly help me.

Regards,

Sujatha Menon

Anyone with experience with cloning using E.coli DH5Alfa and Pjet 1.2 plasmid who can share their experience with me? I've been trying to understand and identify if my clones are showing DNA supercoiling, but it's been very difficult to find out about it. Plasmid linearization is a rare subject to find (at least where I looked). Thank you in advance for your contributions.