Polymerase Chain Reaction - Science method

Hi every one, I our Skin Lab research, we focus in Pemphigus Vulgaris diseases, specific the association of ST18 gene with PV. We read the article "Etesami, Ifa, et al. "The association between ST18 gene polymorphism and severe pemphigus disease among Iranian population." Experimental Dermatology 27.12 (2018): 1395-1398.‏" We look at supporting information on websit in paper, especially we look for photo of Gel electrophoresis of tetra-ARMS PCR results, we want to know how the running profile of electrophoresis looks like in gel when there is/ no the rs2304365 SNP, that means what is the number of binds we are supposed to receive especially using this PCR method? Hope to get help and usefull information:)

Hello,

when is the light measurement taking place in qPCR? during the annealing or during the extension?

Thank you

I didn't design my primers that produce A overhang. And I have to do cloning. I read that I can use dATP and nonproofreading Taq to produce A overhang. Can I use this method to add an A to my PCR Product. I assume that My PCR Product is not blunt ended. Coukd you please help me about the isue?

I am genotyping a T-DNA mutant in Arabidopsis and I get a band in the T-DNA reaction for wild type (Col-0). I've run this reaction four times now and used different wild type samples but still seen the T-DNA band. Has anybody experienced this before?

PCR reaction conditions:

Initial denaturing at 95C for 3min

Denaturing at 95C for 30sec (35 cycles)

Annealing at 52C for 30sec (35 cycles)

Extension at 72C for 1min (35 cycles)

Final extension at 72C for 10min

W=Wild type, T=T-DNA

Dear Professors and colleagues

Recently I started to use Takara In-fusion system for cloning of my Gene of target to vector using inverse PCR technique.

But unfortunately, I got an unclear PCR product.

I would like to hear your precious opinion for my study.

Thank you in advance

Our lab is doing Genotype of T0 rice, those plants are regenerated through tissue culture as well as in planta transformation. All the time(Except 1-2 times) in genotyping we are getting bands in every samples in PCR with wild type.

FP= Tm-64.1, GC%-40

RP= Tm-59.2, GC%-36.3

We have used annealing temperature in PCR is 47.6 (This we checked in Gradient PCR with Plasmid DNA) This primer designed on the flanking site of gRNA region and Amplicon length is 281.

We have checked different different things to troubleshoot this problem those are mentioned below

1) Genomic DNA isolated from different methods then used for PCR

2) Each time used new aliquot of Water, PCR premix and primers

3) Prepared new chemicals so many times for Genomic DNA Isolation.

4) Used Different primer set.

5) Used pipette from other labs

6) whole Experiment did on different lab also

I am doing PCR to add a (GGGGS)4 linker at one end of a gene. After amplifying the gene with suitable forward and reverse primer, I got an excellent band on the gel, eluted it from the gel, and checked the agarose gel again. I used the same (the eluted sample) as the temple to amplify with an old forward and new reverse primer having 10 bases of linker sequence at their 5' end. I got an excellent band on gel again, eluted the same, and checked on the agarose gel. But this time when I used elutes sample for PCR with the same primer set, this time no band was observed on the gel. I repeated the PCR with the dilution of the DNA temple, changed the PCR master mix (Takara), and added DMSO to the master mix.

I have amplified a gene fragment and want to send it for sequencing however, its concentration is 25 ng/ul. Is that concentration fine for sequencing?

I made my ligations using pGEM-T easy vector system I, I normally used PCR products (product for TA cloning-Taq DNA polymerase and Control Insert DNA (promega like a ligation control) for the transformation I used uncut plasmid like I control. But after I have plated bacteria and left them in 37C incubator overnight I do not have colonies just find circular water bubble in my LB agar with amp (PCR products and Control Insert DNA) but in my transformation control I have colonies. this demostrated that transformation process is fine and my cells are competent. Is possible that reagents of this lot it is degraded? T4 enzyme for example. I repeat many times following the insert kit protocol, What is causing these water bubbles?. When I prepared my plates with LB media I avoid the bubbles all the time.

I am performing qPCR on drosophila mtDNA. Because of the nature of my work, I use two separate primer sets. With one of them, the qPCR works as expected - good amplification with mtDNA and nothing/ very high Cq primer-dimers in NTCs.

With my second set of primers, I continually get amplification in the NTCs that is at about the same amplitude as the template samples, and have identical melt curves. I have tried using new primers, new readymix, and new water. I have also bought new primer stock, but still have this issue. I have used these primers in PCR and they have worked fine when verified with gels (PCRs are without the readymix, that is the only difference - I make up my own solution).

Because the first primers work fine, it seems like it doesn't seem to be a technical/environmental issue, or contamination of the water or mastermix. The only difference is the primers which I initially thought may have been contaminated, but given new stock solution yields the same results and PCR without the readymix is also fine, that does not seem to be the case.

Is it a thing for primers to be incompatible with certain readymixes? If so, why is that? I haven't found any explanations in my searches.

Hello,

I did PCR for mouse genotyping; the Ct difference for around half of my duplicate samples was greater than 0.5.

I cannot find what the problem is.

If someone could assist me with this issue, I would be very appreciative.

In my research, I performed PCR to identify a marker linked to the R-gene in my samples. I experimented with various primers and finally achieved my desired band size through gel electrophoresis. However, I have since extracted new DNA and attempted to identify the same R-gene-linked marker in similar samples using the same primers and PCR conditions. Unfortunately, I am now encountering a different image with the presence of smear bands. To aid in understanding, I have attached a comparative picture.

Thanks in advance.

We are comparing different DNA extraction methods and we use qPCR for analysis. We conducted PCR on 2 amplificators, but they had pretty different results. For example, one of the methods had 30 negative and 10 positive samples on the first amplificator, but only 4 negative samples on the second one. Moreover, it had the highest Cq value on the fisrt one, but the lowest on the second one. What could be the cause of this? We used the same program and reagents for both amplificators.

I have the following question: I have a nested PCR protocol that uses a chemically modified hot-start polymerase. However, in the lab, I have a hot-start polymerase, but bounded to an antibody. The purpose of the PCR reaction is to amplify a specific fragment of a viral genome, which will subsequently be sequenced by the Sanger method. The question is, can I use the polymerase I have, and will the antibody that blocks the polymerase activity before the appropriate temperature, interfere with the subsequent sequencing analysis? Thank you for your answers!

To have a sure positive control for a PCR, I have ordered an oligo containing the cognate sequences of the primers (20 nt each) separated by 150 of random DNA; before and after the binding sites, I left five nt so the overall length is 200 nt.

When I ran the PCR, nothing was amplified.

Could it be that 5 nt upstream/downstream of the binding site are too few for the enzyme to work properly?

Also, could it be that having both cognate sequences on the same strand is a bad idea because the enzyme would collide releasing only small fragments?

Hello All

I have a question regarding real-time (RT) Primers Optimization. We usually perform gradient-PCR with standard PCR conditions for primer optimization for qualitative PCR. But for RT-primers optimization, what changes should I make? I put on standard PCR conditions but I got non-specific bands.

Can anyone help me in this regard?

Hello everyone,

I would like to ask a question to manage my experiment. To decrease the budget of my experiment, I have been searching for designing miRNA primers for RT PCR. I could find just a few sources about it. Is there anyone who applied it before or can suggest some articles or programs to design the miRNA primer for RT PCR?

Thank you so much.

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?

I performed PCR for genotyping but I could not amplify one of the sample while other samples is amplifying just fine... even I put + and - control, the + ctrl is amplifying well... what could be the reason?

Regarding sex determinant in birds by Real time PCR. How can do it?

Hi

I'm doing asymmetric PCR to amplify ssDNA, using F:R=1:10 ratio of primers.

I already optimized the PCR cycle, annealing temperature of my PCR conditions, but when I detected my product with 1% agarose gel, ssDNA band detected higher than the dsDNA band. As i know, ssDNA has to be lower than dsDNA because they migrate faster. Stranger thing is dsDNA band from the asymmetric PCR product showed higher than the normal (F:R=1:1) PCR product band. My target product's size is 185bp.

Does anyone tell me what is wrong?

I'm using 1% agarose gel, 70V 30-40min (same as DNA ver. agarose gel - I confirmed that electrophoresis gel condition for DNA did not affect to ssDNA detection)

Please find below some of the details related to the study:

1. Total no of samples: 640

2. Organism genome size: 500 mb (in related species)

3. Restriction enzyme pair: SbfI and MSpI

4. Size selection fragments: 250-600 bp

5. Platform for sequencing: NovaSeq PE150 (120 G raw data per sample)

6. Multiplexing: 48 adapters X 12 PCR index

Looking forward to getting some help here

I am having issues resolving PCR inhibition in rhino dung samples for a metagenome sequencing project. I used the DNeasy Blood and Tissue Kit to extract gDNA (with expected low yields, but DNA present all the same), and have attempted to clean extracts amplify the 18S region using NEB's Q5 HiFi 2x Mastermix. I know that the primers themselves are robust, the positive control looks great, and all fecal samples EXCEPT for rhino amplify well! Cleanups/Optimizations that I have tried: 1) Monarch Kit (best Nanodrop 260/230, 260/280 values), 2) Double extractions using Qiagen's DNeasy columns, 3) Dilutions (1:5, 1:10, 1:20, 1:50, 1:75), 4) Optimizing PCR by adding 5% DMSO and 5M Betaine, 5) Zymo's 1-Step PCR Inhibitor Cleanup Kit.

Help!

I have Tet and Fam labelled hybridisation probes pairs that have successfully worked on a ddPCR platform. Transferrring to real time PCR using the QS5 machine I can no longer detect the tet labelled probes (or very weak signal). The samples are intact as tested with GAPDH probes (Vic label) .The QS5 is not optimised for tet but I have calibrated the machine as per instruction manual.Anyone had any success with Tet probes on the QS5 or similar?

I am trying to PCR amplify bacterial 16s ribosomal RNA gene (from different bacteria). Can I please know an accurate universal forward and a reverse primer sequence for this purpose?

It is known that it prevent mispriming and unspecific amplification . can anyone explain the exact mechanism.

Leishmania

Hello fellow researchers, I did a simple CUT&TAG for H3K27me3 in HEK293T cells. I checked my results using PCR. If you look at this picture that I attached, I first thought I had untagmented DNA bands in my samples. However, this doesn't explain the band in the IgG sample which is the first lane after the marker. Because, if it's untagmented genomic DNA, it's in every sample including IgG regardless of antibody treatment. Any insights for troubleshooting will be helpful.

We are looking for RT PCR kit that we can use to amplify Telomerase Reverse Transcriptase(TERT) and then conducts a qualitative PCR.

Lysis buffer for RNA extraction TERT

Specific primer/probes (oligo (dT) hTERT primers)

Master mix (reverse transcriptase, dNT’s, Recombinant RNasin Ribonuclease Inhibitor, reaction buffer

In my experiment, i have used a specific primer for identification of E.coli o157 however, the positive control samples showed good results on gel electrophoresis but the random collected samples showed no results. the collected samples showed pink colonies when cultured on specific chromogenic E.coli agar the same as positive control.

Normally its a common scientific notion that as we decrease the annealing temperatur, it’s likely more primer dimers are formed. In this picture, I have observed something differen. As I increased the annealing temperature, primer dimer formation increased and exact amplicon size band became lighter

pcr: Gradient (extremely useful if you are unable to amplify the gene)

amplicon size: 968 bp

primer dimer: 200-300 bp

might be you are thinking why I am saying primer dimer at 200-300 bp site? I give you answe, first wanna listen fron you

Is there any significance in term of sensitivity/specificity?

We have 150+ glycerol stocks of unknown microbes (from soil) that we need sequenced and we have tried sending them off for direct colony sequencing using universal 16S primers and it failed (even the known ones we sent in) so I'm stuck. We have considered doing DNA extraction first but that would be very time-consuming (and we need these sequenced ASAP) so we're considering doing colony PCR (using universal 16S primers) and PCR cleanup and sending that off for analysis but we're having trouble producing clean bands. Any suggestions? It is possible that some of these are fungal so can I combine both primers together? TIA

Hello, can anyone please describe to me an effective way to amplify a plasmid backbone. When I use my primers, I get a lot of non-specific priming and do not get a strong enough band (if any) at the position I should, but I do get strong bands at a position much lower. I have tried doing a touch-down PCR, and I am using Q5 taq.

I am inserting a gene several times into a plasmid to make a large construct, so any tips for the future would be awesome, as I will have to amplify a new backbone several times.

Thank you :)

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?

Hello,

I'm doing a SOE PCR. As soon as the PCR is done, I run electrophoresis of the samples. The gel shows the marker but no sample, as if I didn't load the sample with the loading buffer. But then, I run again the same samples in the same electrophoresis chamber with the same conditions and it shows results. What can it be? This happened to me 4 times, and I'm losing my mind. I couldn't find any answers.

We have a certain protocol that we follow in lab for a PCR test that we do with our DNA. We have been doing this same protocol for the past 2 years. For the first year, it has worked just fine. Recently, however, we have begun to see what looks like contamination even in our water. No matter how often the water is replaced or anything, the band will often appear. We are using the same conditions as we have been for the past 2 years. Has anyone ever noticed a decline in the performance of a PCR machine, or differences when they run PCR machine versus another?

We performed qPCR analysis using SybrGreen dye, where the template was gDNA, on the Applied Biosystems 7500 Fast Real-Time PCR System (software ver 2.3). The reaction conditions were optimized using standard PCR conditions (denaturation at 95°C for 60s, annealing at 58°C for 30s, elongation at 72°C for 30s). First, these conditions were tested. We also tested the default program for SybrGreen (2 step, 60°C annealing + melting curve, photo1,2) and many other combinations each time, without obtaining any curves (photo3). I know that the reaction itself, reagents and temperature profile is well optimized because after the reaction, we made an electrophoresis of the product (photo4).

Do anyone have experience with this software? Is this software bug (do you press any extra option?) or do I make something wrong? Additionally, is it possible to preview the amplification plot such that it is presented on a linear and not a logarithmic scale during reaction? I will be grateful for any tips.

How can I know the product size in base pair, while I don't have positive control and the reference paper did not mention the expected product size?

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 set up a bulk PCR reaction of 200 µl volume. I got the band of expected size (about 900 bp) which was to be extracted (run on gel made with TBE buffer). This worked fine for the first 2 times. However, the same setup is not working anymore. The PCR product obtained showed a very faint band of expected size (run on gel made with TAE buffer). However, after gel extraction and PCR purification, when the eluate was run on gel, there was no band.

What could be the reason for this? Did the PCR amplification not work? And if so, why did it not work when it had initially worked fine with the same conditions?

Hello,

I have a question regarding PCR. I am trying to do a site-directed mutagenesis on my plasmid.

I use fresh PCR water, change tips after adding something into one tube and I also let all the components thaw on ice before using them. I first add PCR water, then my plasmid template, then the primers and at last the MasterMix. I tried different annealing temperatures but only one time it worked. After repetition the band was lost again. Still my band is missing and I am quite confused what the reason is.

You can only see one band at a different bp-amount on the gel and also see some band for primer dimers quite faintly.

What could I be missing? What could I change?

Dear all,

I was creating a site-saturation mutagenesis library for one site using inverse PCR based on the paper (Zheng, 2004). PCR reaction was performed using Q5 or Phusion DNA polymerase and the host cells were E.coli H5α competent cells. The plasmids extracted from transformants were sent for sequencing, which revealed that there were multiple undesired forward primer sequences successively inserted next to the primer in almost all the resulting plasmids. Did anyone have such experience or could someone offer some ideas? Any help would be highly appreciated.

The forward primer: GATACCGCACCGnnkTATGGCATGGGTCTGAGTGAAC

The reverse primer: GACCCATGCCATAmnnCGGTGCGGTATCAAAGTAGC

Best regards,

Hi every body, we have seaweed samples extracted using CTAB protocol, DNA quantity and Quality was good. We did PCR for the samples using ITs ribosomal DNA and rbcl marker. Positive control was used from the same samples extracted before. We don't have a PCR product for our samples but we have clear band for the positive control. What may be the reason for the PCR failure in this case?

This a ligation PCR, which I'm doing it to check if my ligation worked or not. I am using two primer pairs with very similar annealing temperatures. Ideally, if the ligation worked it should answer for both primer pairs. The expected size for the first primer pair is 387bp and the second is 1kb. The template used for the PCR was the different ligation mixes. But both primers had all the ligation mixes. For example, a 1:3 ligation mix was used as the template for both primer pairs. I have thin bands around 0.5kb for the first primer pair indicating the ligation may have worked. But I have no bands for the second primer pair. I am not sure why this is the case. I can only think of two reasons: the template concentration was not enough (cause after all, the second primer is a longer amplicon) or the time I ran was not enough. I had put 45 sec for annealing, assuming that it takes 30 sec for 1000bp, so I should be good anyways. I'd appreciate any help trying to understand what happened! Thank you!

I have primer designed for conventional PCR and would like to use it for qPCR / RT PCR by adding probe as one reagent in master mix if that is possible.

Hello everyone,

for a couple of weeks now i try to realize a PCR amplification of DNA extract from coral of the species Pocillopora.sp using ITS2 primer (internal transcribed spacer region 2)

for around 3 weeks now, i keep trying to realise the PCR but fail at getting negative Blanks for absolutly uknown reason.

the last PCR i did today just confuse me at the maximum.

here is the link to it https://drive.google.com/file/d/1kspwqAmiBQ5uAwCFNDg_x8jJa2x90qUq/view?usp=share_link

i won't describe everythink i did but i have been extremely carefull to clean everythink, use mask, lab coat and so on. I also used a laminar hood and placed all the material i used under an UV light for 30 minutes.

the first row of the gel is only blanks. i have prepared a master mix and haven't touched my sample at all. i just placed the master mix in one row of the plate and closed it with capes right away so there is absolutly no posibility for cross contamination.

so from that i just came to the conclusion that the contamination must come somehow from the material i use, even tough i take great care of cleaning everythink.

The second row goes as follow (sample/sample/sample/sample/space/blank/blank/blank/blank)

again, all my blank came out as strongly positive but then i don't understand why some of my sample came out negative since the first row tell me that the material is contaminated.

if my material is contaminated, although my sample is negative it should come out as positive

i am completly out of idea right now, i have tried basicly everythink that existed but can't figure out the problem.

if any of you has ever had a similar problem, i would really appreciate the help

Hello,

I am trying to use the Quiaxcel advanced capillary electrophoresis system for fragment analysis of PCR products that have been cut with a restriction enzyme.

I dissolve my ladder (size marker) in PCR buffer, Restriction enzyme buffer (containing BSA) and EDTA in similar concentrations to the ones in which my samples are dissolved. However, doing this the resolution of the ladder is not clear and I can't see the expected peaks in my samples.

Does anyone knows if any of the buffers I am using influence the electrophoretic run, or does anyone that has used the machine before has any suggestions?

Thanks a lot!

I am trying to replace a 20 bp region with a different sequence of same lengthin a 10kb plasmid. I designed overlapping primers. However after PCR I am only getting the template. At first I thought I was getting amplification but on Dpn1 digest everything is getting cleaned up. I tried both multiple ramp and single ramp. Did multiple temperature gradients. But always the same issue.

I am quantifying level of gene expression using qRT-PCR. I want to know at what stage is best for normalization of samples. Is it at the Reverse transcription stage (RNA normalization) or PCR stage (cDNA normalization) or both stages and the reason behind.

Thank you

I had gel eluted a PCR product whose concentration according to Nanodrop was 16.5ng/ul. 20ul of this PCR product was treated with 0.5ul of FD Dpn1. Hence, the yield of PCR product in this 20.5ul reaction mixture was calculated to be 16ng/ul.

Now, 1.53ul(1.23ng/ul) was used to treat with T4 PNK(0.4ul) in a total reaction mixture of 20ul. PNK was then purified using spin filter and column and DNA was eluted using 15ul of Elution buffer.

Reading on nanodrop is showing A260/A280 of 4, which does not indicate the presence of DNA. Hence, How do I calculate the concentration of DNA after PNK treatment?

I am conducting Avian Influenza virus detection by real time rt PCR.

I did 4 target AI genes and 4 primer probe sets for PCR.

(H7 HA, H5 HA, NP and M target gene)

I run real time 4 time individually for that 4 target genes.

The result came out non-similarity.

(For Example,

In Sample no 1, H7 HA and H5 HA amplified (positive) but not amplified in NP gene and M gene .

In Sample no.2., result came all 4 gene were amplified.

In Sample no.3 NP gene and M gene and H5 HA gene came out positive result.)

How can I understand the nature of antigenic protein surfaces of virus and detection system of real time PCR.

Even though the same sample and same virus, can different results by using different target gene?

Hello,

I have a question regarding qPCR efficiency calculation.

Is it correct to talk about PCR efficiency if I perform a serial dilution of sample that containing an unkwon concentration of the viral particles?

I usually read that the serial dilution should be done from a known concentration of the extracted DNA. But I have stool samples where the amount of virus is unknown and the dilution was made from the sample stock. Afterwards the DNA was extracted and PCR was run.

When I calculate the slope of standard and the efficiency. Can I then talk about PCR efficiency?

Thank you for your feedback!

How can I get my final PCR concentration to be 20ng/uL within a final total 25uL volume, where 2 uL of this final volume is from my DNA template while the other 23uL is from the mastermix (buffer, dNTPs, primers, etc. have all been calculated for a 2uL DNA template PCR reaction).

I used the nanodrop to get the concentration of multiple samples which they all had different concentrations (167ng/uL, 263ng/uL, etc.). I understand how to use C1V1=C2V2 to dilute these concentrations to similar concentrations; however, each sample must use 2uL of the DNA template into 23uL of the mastermix, and I don't know how to get the final concentration of all the PCR samples (must all be the same due to I am using band intensity analysis with gel electrophoresis) to be 20ng/uL?

For example, if I took all my stock DNA samples and diluted them into 20ng/uL, I would need to take 2uL of this 20ng/uL DNA and put it into 23uL of mastermix. I understand all the samples would be the same concentration but it would no longer be 20ng/mL because it got diluted again. I cannot adjust the volumes of either the DNA template I use (2uL per sample) nor the mastermix (23uL per sample), the only thing that I could adjust for is the concentration of the DNA diluted stock that I am pulling my 2uL of DNA template from.

What should I dilute my stock DNA concentration to so that I can get 20ng/uL DNA within a total volume of 25uL where I MUST use 23 uL of mastermix and 2uL of DNA template?

Dear Colleagues,

I wish to perform a genotype-phenotype correlation study in a family with a known mutation in RPGR-ORF15.

However, the RPGR-ORF15 region is very tricky because it contains GC repeats and thus its not possible to amplify with standard PCR conditions.

Special protocols are needed for both PCR amplification as well as during Sanger sequencing.

Therefore, I would greatly appreciate if anyone could suggest me a commercial facility where I can send my samples for screening the specific mutation located in the RPGR-ORF15 region?

Number of samples to be screened = 05

Many thanks,

Atta

My PCR reaction as follows

DNA Template - 10-100 ng

10X PCR Buffer - 5 µl

50 mM dNTPs - 0.5 µl

Primers (100-200 ng each)- 1 µM each

Water add to a final volume of 49 µl

Taq Polymerase (1 unit/µl) -1 µl

Total Volume - 50 µl

denature 94c for 2 minute then 10 cycle for 94c 30sec and annealing temp 71c to 61c then 15 cycle 61c annealing 72 c extension and final extension 72c for 4minute

Could you please indicate me a source, or any useful information for where can I find the genome copies of 16s RNA of pseudoramibacter alactolyticus. I need this information for several other bacteria because I need to perform standard curves on PCR.

Please take under consideration that I am totally novel at this issue and I need help with my research

Thank you

How can you use PCR to amplify and detect specific DNA sequences in crop samples?

I am trying to use overlap extension PCR to combine 2 linear PCR fragments around 1kb each. I amplified both fragments with overhanging primers with a 20 bp overlap between the two fragments. When I do overlap extension PCR, I just get amplification of the individual PCR fragments. I am doing a PCR reaction for 15 cycles without the primers, and then adding the primers that flank either end of the combined product for another 15 cycles.

Does anyone have suggestions for troubleshooting? The overlap region between the two fragments has a TM of 54, and the primers have TMs of 74 and 78. For the overlap PCR reaction I tried an annealing temperature of 50 and 55, and for the extension reaction I have tried annealing temperatures from 55-70.

What is the amplification program used in the thermocycler for PCR of the Sox C gene, and what are the specific temperature and time conditions for each cycle of the amplification program?

Hi everybody,

I have tested a kit for cell free protein expression (Next generation cell free protein expression kit, wheat germ CFPS 700) from Merck and I didn't get the expected yield for protein production.

In the procedure of this kit you have to prepare DNA template by a game of several PCR, then in vitro transcription is realized from PCR template, and finally cell free translation using wheat germ extract.

All is good until transcription (agarose gel checking)

But after that the protocol is a mRNA purification using amonium acetate salt and ethanol.

I think these step is the problem because I loose a lot of mRNA.

Can somebody tell me if this step is necessary or if I can try to translate without mRNA purification? Or else, is there another methode for mRNA purification, that preserve its quality for the following transcription (the kit exclude phenol, trizol or ammonium sulfate purification that rendered mRNA unsuitable for translation)?

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 am getting 3PCR products of different sizes when amplifying off a plasmid, and I think it is due to unintentional primer binding. I am amplifying a trimer off the plasmid, and the 3 sizes correspond to the monomer, dimer, and trimer. My primers bind to the parts of the plasmid flanking the trimer, so they each only bind to the plasmid once. The primers have overhangs so the PCR product can be used in Gibson Assembly, and I am thinking that the overhangs may be close enough in sequence to bind somewhere on the trimer. Are there any tools to check primer specificity, or do you have any suggestions about what else could be going wrong?

Hi, I would like to ask if anybody has positive experiences with single primer PCR ? Can you recommend me any proven protocol of this type of PCR ? Thank you for all recommendations. Bohuš

Could anyone recommend Mycoplasma test to be used in a cell culture laboratory and that would not need the access to PCR?

Thank you!

I am not looking for a PCR calculator. There are many PCR calculators that claim to calculate the melting temperature "Tm", but this is false. They calculate polymerase-dependent values of interest for experiments involving polymerases.

Instead, I would like to calculate the melting temperature, as it would be revealed by a melting curve experiment. No polymerase is involved in this experiment, and the value does not depend on polymerase selection. It depends only on sequence and buffer composition.

Do you know of a software that does that well?

1 I got both outer and inner band for wild type on temperature but no band for mutation now i am getting no any band on the same conditions on which already got band while optimization.

2. if I don't have any positive sample of mutation then how can I confirm about primer specific for mutation is working or not!