PCR Analysis

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Questions related to PCR Analysis

What should I do if the forward primer binds at two sites?

I wanna do a PCR with a forward and a reverse primer. Now I have the problem the forward primer is binding at two sites of the DNA. I can't change the forward primer because of my enzyme of...

07 July 2015 5,904 9 View

Will the 3' polyA tail be washed away if I purify PCR product with beads?

I want to clone a 3kb fragment. I use Phusion first to increase the fragment, run a gel, cut the size I want, purify the gel, use Taq to add 3' poly A tail, purify the PCR product, and then do the...

07 July 2015 9,339 5 View

Does my no template control amplification indicate necessarily contamination?

I'm performing some qPCR reactions. My experiment is only about absolute quantification of genomic DNA from some samples. My no template control sample is showing flurorescence at cycle 33. The...

07 July 2015 8,230 2 View

In my first trial in Methylation specific PCR, i got non-specific bands considering the methylated and unmethylated control, Why this happened?

My PCR Protocol was: Hot start: 95 °C for 10 min Denaturation: 15 s at 94°C, Annealing for 30 s at 61°C, Extension step for 30 s at 72°C; Final extension for 10 min at 72°C; A hold at 6°C. For 35...

07 July 2015 285 3 View

What are the best conditions when using PuRe Taq ready-to-go- PCR beads?

I'm using the ready to go PCR beads for the first time. The protocol recommends a final volume of 25 uL. I tried using 2 uL of primer mix (1 uL of each primer, at a concentration of 5 uM, diluted...

07 July 2015 3,368 1 View

Can anyone help explain double bands in PCR?

I am getting two bands below 100 bp in addition to the gapdh amplicon . What might be the reason for these bands? Lane 2- positive control lane 3-6 - samples lane 8- negative control

07 July 2015 4,112 6 View

What is the best method to concentrate PCR product after pooling several samples?

Hi, I need to pool about 50 samples to send for sequencing that will obviously leave me with too great a volume as the DNA I am dealing with is at very low concentrations. Having pooled them I...

06 June 2015 8,602 12 View

Why can't I re-amplify error-prone PCR products?

We have tried error-prone PCR with GeneMorph II Random Mutagenesis Kit and another kit from a Chinese supplier. Both kits worked fine in producing a specific product (single band on gel) of...

06 June 2015 8,231 3 View

Any advice on the AFLP amplification step?

Hello! I want to know the genetic variability of C. acutatum collected from Korea So, I am working on the AFLP analysis of C. acutatum. first, I conducted the pre-amplification step and...

06 June 2015 6,578 6 View

How can I eliminate 2nd band on PCR?

I am trying to optimise my PCR, I know the Fungus is Aspergillus brasilliensis. I am using ITS1F and ITS4 primers with Tm of 56.6 and 61.5 respectively. I have changed cycles, annealing times and...

06 June 2015 9,674 2 View

Are faint bands in PCR real or not?

I'm having some trouble with genotyping for this one gene & hoping to get some insight! Our primers have an expected product size of ~600bp. As you can tell by the picture of the first run...

05 May 2015 183 2 View

What is the best way to find the annealing temperature for the second step of PCR?

I have primer set that has annealing temp. 60-61degree centigrade and GC content 55-60%. First step always gives good band the second step of touch down PCR gives non consistent bands under same...

05 May 2015 1,053 9 View

Is there any method to detect mutations caused by Taq Polymerase with less proofreading capacity and low fidelity?

I have used a Taq Polymerase with less Proof reading and fidelity. I have a doubt that some mutation may be occurred during the PCR. One drawback is the polymerase doesn't have 3'-5' exonuclease...

05 May 2015 8,576 4 View

Can anyone help in standardizing the conditions for the amplification of a 120bp DNA fragment, with approx 26 bases 5' overhang in the forward primer?

I am doing an overlapping PCR to ligate 3 DNA fragments. PCR for the first two fragments have worked . I am troubleshooting PCR conditions for the third fragment. This DNA is 120 bp long. The...

04 April 2015 4,205 5 View

Can anyone help with the absolute quantification Real Time PCR analysis to analyze the ct values obtained and convert them to the expression levels?

I had 10 different tissue samples, of which 5 were normal subjects and 5 were diseased. I have ran a AQ RT PCR for my gene along with GAPDH and I have the ct values. But how to analyse expression...

04 April 2015 2,989 7 View

Can someone help me to quantify the mRNA levels of a particular gene in the G1, S and G2/M phase of the cell cycle with rt-PCR?

My question is regarding rt-pcr: I am trying to quantify the mRNA levels of a particular gene of my interest in the G1, S and G2/M phase of the cell cycle. However as i have read a housekeeping...

04 April 2015 6,754 3 View

Is omitting outliers allowed in qPCR?

I am validating my primer which will be used for relative quantification by doing a serial dilution. For 2 of my dilutions I have an outlier that is affecting my R2. I know an acceptable R2 is...

04 April 2015 9,727 6 View

What is the best stastical test to analyse methylation specific PCR results?

Through methyaltion specific PCR we got three results: methylated, unmethylated and heterozygous. The question is, what is the best statistical test and how do I analyze the data. Thank you.

03 March 2015 1,403 3 View

In the case of presence of unspecifics bands in a PCR product, can a dilution improve the quality of sequencing ?

I maked some PCR of rpoB gene (Mycobacterium tuberculosis). I obtained my interest fragment of 411 bp together with some unspecifics bands that disturb sequencing step: the qualities of...

03 March 2015 6,513 5 View

Any suggestions/ recommendations for identification of Mosquitoes from Southern France?

I'm doing an Mres project on avian malaria and want to include analysis of mosquito prevalence/ emergence during the field season in order to include the full malaria parasites life cycle. I will...

03 March 2015 1,877 24 View

Will somatic DNA influence the bacterial DNA when doing downstream PCR analysis?

Hi, as i want to extract bacterial DNA from mice tissue, but the final solution also contains both bacterial DNA and background DNA (mice tissue DNA), Will the somatic DNA influence the bacterial...

02 February 2015 4,023 8 View

Has anyone used standard Polymerase in a long-RT-PCR?

I'm currently trying to achieve a (around) 1kb amplicon from peripheral blood mRNA but don't get any resuts, and i'm not capable of buying the long-pcr kit. Any advise or experience is...

02 February 2015 6,123 3 View

Is QIAamp DNA stool kit suitable for DNA purification from faeces for Balantidium coli PCR analysis?

I have around 100micrograms of whale faeces. The sample is positive for Giardia and Balantidium coli and I need to do phylogenetic analyses for both parasites. For Giardia, I can use the QIAamp...

02 February 2015 2,624 4 View

What is the probability of replacing U nucleotides with digoxigenin during PCR amplification?

Thank you.

02 February 2015 4,434 1 View

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