How can I ensure that my sequence data includes the region of interest?

We have a PCR assay whose product is later sent for simple DNA sequencing. Because we were restricted with our primer design (high A-T content) we were only able to design 1 set to use for both...

04 May 2018 5,369 14 View

How can I get rid of nonspecific band in product?

Hello, I am designing a Taqman assay and have used ABI's Primer Express software to design the primers and probe. I've also had a bioinformatician check that the sequences are in conserved areas...

02 March 2017 8,726 2 View

What effect does low 260/230 have on downstrain applications, like RT-PCR?

There seems to be salt in my RNA after extraction. I proceeded to perform a cleanup step and while the quality (260/280) improved, the 260/230 remains low. I do not have more sample to...

04 May 2016 5,396 6 View

I have a large unexplained band in my gel. Can someone help me troubleshoot, please?

31 December 2015 1,465 7 View

What could the unexpected band in my gel be?

I extracted RNA from a positive and 2 negative samples. I then synthesized cDNA and performed RT-PCR. I inspected the amplification plot and melt curves for these. The positive sample came up on...

08 September 2015 7,530 15 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...

03 April 2015 9,612 6 View

Do all 5 dilution points need to be amplified to get an accurate primer efficiency?

I am trying to find the efficiency of my primers by doing a serial dilution of my sample, but the last 1 or 2 dilution points fail to amplify (or my triplicate points don't fall in the same Ct...

02 March 2015 7,551 4 View

If I decrease the extension temperature in my real time PCR, will I increase the time?

02 March 2015 494 10 View

What do I use to design primers, mRNA or genomic sequence?

02 March 2015 1,015 13 View

Can I validate (check efficiency) primers using gDNA?

I will be using the primers for gene expression using RNA samples, but can I validate them first using gDNA or does it have to be with RNA, or both?

01 February 2015 3,851 16 View

Does any one know how i can prepare liver tissue with transmission electron microscope with good fixation?

i have problem in preparation of liver tissue with bad cell organelles and how i can adjust osmolarity

03 March 2021 8,890 1 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

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

01 March 2021 5,311 1 View

Expression cloning by using cDNA,do I need to modify the cDNA first?

I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...

28 February 2021 5,440 3 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