PCR - FAQS.TIPS

More Bushra Javaid's questions See All

Primer designing from reverse translation of protein sequence

Hi, I am interested to design primers for my protein of interest (3' RACE). I have N-terminal amino acid sequence of protein. When I reverse translate that protein, codon usage table is required...

06 July 2017 3,028 4 View

Hi, I need help to defat samples before protein extraction. Should I treat seed samples with hexane only or fresh samples also need treatment?

Hi, I am going to freeze dry samples prior to protein extraction for transport to some other destination. As seeds contain so much oil these are treated to defat them before protein extraction.I...

03 April 2015 6,476 6 View

To which extent should I concentrate the methanolic and the aqueous extracts?

I am preparing methanolic and aqueous extracts of some plants. After evaporating solvent by rotary evaporator small volume e.g 20 ml is left. I want to ask to which extent I should let it...

01 February 2015 9,765 2 View

nested pcr

hi, i am doing nested pcr these days but my outer primers are not giving bright required band but other non-specific dense bands. I am using master mix recipie for 25 microliter pcr reaction...

09 October 2010 6,666 2 View

Nested pcr

Hi, I am doing nested pcr. I have completed round of pcr with outer primers now i am going for nested round of primers should i use pcr reaction as such or dilution of pcr reaction with outer...

09 October 2010 2,092 3 View

How do I increase the yield of my PCR product?

Hello, We would like to increase the yield of our PCR product. We are running a series of PCR reactions that is targeting ~1.1kb sequence. We begin each reaction with ~400pg of template DNA...

02 March 2021 4,029 3 View

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

I am going to have 3 different probes in my qPCR work that I am going to do. But I realized that the machine we have in the lab is a Rotor-Gene Q 2plex HRM Platform, saying it has green, yellow,...

01 March 2021 8,544 1 View

How to address this issue in the analysis of Real-Time PCR results?

To dear Researchers, I was analyzing a series of concentration for estimation of Real-Time PCR efficiency. The concentration was 1:10. I used MS-excel to evaluate Slope. The result of slope was -8...

01 March 2021 8,683 4 View

No title

Does anyone have the experience of using Taq Man probes in the QIAGEN Rotar- Gene qPCR machine?

01 March 2021 5,311 1 View

MiRNA qpcr problem?

Dear All, mirna primer showing some problem in the melting curve? any idea why? As attached is the melting curve. The forward sequence is obtained from miRBase and reverse primer is universal.

28 February 2021 5,008 4 View

Runs of nucleotides in Sanger sequencing?

I performed site directed mutagenesis, transformation, and then I sent out plasmids for Sanger sequencing and found out that there is extension of DNA just before the stop codon. I am not sure...

27 February 2021 547 3 View

Can thermal cycler machines be problematic during PCR?

Hi, my question is about the heating of thermal cycler machines and I hope some of you had experienced a similar thing previously. There are two thermal cycler machines in the lab(BioRad) and for...

26 February 2021 4,777 4 View

Calculating volume of DNA required from DNA concentration?

Hi I am a bit confused. They are asking me to find out the volume of DNA required in ul (a total of 30-100 ng for genomic DNA) from the DNA concentration in the nanodrop reading which was 404.8...

26 February 2021 5,029 2 View

Has anyone used the Illumina ITS1 primer sets?

Hi everyone, Illumina provides a list of primers to amplify with high taxonomic coverage the ITS1 region for further fungal sequencing, but I cannot find the exact amount necessary of each...

25 February 2021 6,969 3 View

Why do the PCR bands from the cDNA templates (extraction from the cell after transfection) show different size from the band of the plasmid template?

After transfection with the plasmid ( linearized ) and subcloning of the cell lines, RNA was extracted from the cell and then reverse transcripted to cDNA. When PCR reactions were run to verify...

25 February 2021 5,712 3 View

Alexander Gorbushin

You are contaminating your PCR mix with EDTA.

Try to re-precipitate your primers with ethanol and dissolve again in milliQ water. If unsuccessful, order new primers.

Raunak Shrestha

10X TE buffer is way too concentrated and high amount of EDTA will seriously inhibit the PCR reaction ...!!! Try to dilute the mixture of [Primer+10X TE] to 1X or Try to re-precipitate your primers with ethanol as the previous comment says ...!!!

Netrapal Sharma

hi if your primer not working presipitate your primer with ethenol percipitation than dilute it in miliq water.............