Problem in PCR ? - FAQS.TIPS

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Protocol for extraction and derivitization of fatty acid for GC analyis?

I want to extract my P450 reaction mixture and derivitized for GC analysis. I have read about BSTFA method, but i have no idea about the procedure. Please help me to understand the procedure.

31 December 2018 6,860 12 View

How to purify digested PCR product or Plasmid in more concentartion?

I am trying to purify my Digested PCR product and Plasmid with Promega Kit.The purified product is of low conc.

11 December 2018 2,903 0 View

How to fuse two independent domain?

I have two independent domain cloned in E.coli and i want to fuse them both just to get a single domain by PCR. How to get this by PCR?

09 October 2018 7,125 5 View

How to prepare 2% DMSO soln (v/v)?

I have to conduct assay for enzyme.For this, I have to dissolve my substrate(palmitic acid) 200mM in DMSO having conc 2% (v/v). Please help me .

09 October 2018 2,372 0 View

Facing problem in Cloning?

facing problem in cloning.No colony in transformants plate.

07 August 2018 9,241 10 View

Facing problem in substrate binding with BM3 protein?

During performing assay with BM3 protein (CYP 450),I am facing problem with binding of substrate.I am using Napthalene and Palmitic acid as a substrate.

06 July 2018 1,978 3 View

Facing problem in cloning using Bamh1 and Not1 as restriction sites?

I am using two enzymes BamH1 and Not1 to cut my insert and vector both.Aftr transformation,I am not getting any getting? Do Bamh1 not1 are not compatible ?

05 June 2018 4,541 1 View

Do we need to add potassium phosphate later in tb media?

Do we need to add potassium phosphate later in teraffic broth media ?

05 June 2017 7,302 0 View

Composition of sonication buffer for bacterial cell lysis ?

05 June 2017 6,251 8 View

Do we express our recombinant protein into TOP10 instead of BL21(DE)?

Do we express our recombinant protein into TOP10 instead of BL21(DE)? Is this affects its protein expression?

03 April 2017 7,309 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

Kiran Sreeja Jayan

PCR amplification conditions vary depending on the template, G+C content, primer annealing etc. Most of the time adding PCR additives such as DMSO (3% final concentration) works!

Abhilek Kumar Nautiyal

I am doing same reaction.all the time.Earlier my positive result comes..but now it wont works anymore..

Did you optimise your first PCR with genomic DNA or plasmid DNA? There are many possibilities. What do you mean by saying you have changed the DNA? Did you prepare a fresh one? Any possibility of DNA degradation? You can run it on a gel and see if you still have some DNA?

@ Kiran.I optimized my pcr with genomic dna,at that time all my results are positives.Now i did pcr with same dna,but results are negative.Then i isolate the fresh dna ,again the results are same.Currently i rumn pcr with fresh set of reagents.but the results are same.no bands in gel electrophoresis.Only primer dimers are visible in gel.