In PCR, why do the salts differ for particular polymerase enzymes like Mgcl2 for Taq and MgSo4 for Kod polymerase?

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After purifying protein of interest their is non-specific multiple bands above and below the protein of interest band. Can DEAE method be used?

I am trying to purify a protein and after purification the expression of protein of interest is pretty good but their are non-specific bands below and above the protein of interest. Can anyone...

08 September 2015 2,957 12 View

I had done PCR standardization to amplify my gene of interest of 1.5kb from cDNA. When I repeated the same protocol, no amplification was seen. Why?

I had done PCR standardization using cDNA as template. I did too many pcrs and finally standardized to amplify the gene of interest of 1.5kb from cDNA. I amplified 2 different genes but of same...

10 November 2014 2,919 8 View

I did cloning for phusion protein. I did westerns and getting a band of interest along with a band with low molecular weight, why?

I had cloned my gene of interest in to a vector, which has fluorescent protein tag. I did restriction digestion and did insert release of 1.4kb and fragment release having both gene of interest...

06 July 2014 6,175 8 View

Why am I not able to get a clone of gene tagged with GFP?

As previously discussed, I redesigned the primers. I tried triple ligation, it did not work. So, the gene of interest was restriction digested from the vector containing gene and was amplified...

31 December 2013 604 9 View

I am not getting a desired amplification for new set of gene specific primers designed?

Forward primer: 79.6 degrees melting temperature and Reverse primer: 81.1 degrees. Reverse primer has strong secondary structure and no dimer. I tried pcrs setting with high denaturation temp and...

10 November 2013 2,138 16 View

How to write scientific paper related to clinical psychology in neurological disorders?

I have collected the data and analysed. But writing discussion is little difficult, as I am looking for pathology in brain for psychological symptoms. Please suggest me as to how the discussion...

08 September 2013 9,551 5 View

PCR reaction is not working and not showing any amplifications, while positive control shows. I changed buffer, Mgcl2, dNTPs mix, water. Why is this?

I have been doing genotyping for a mouse model. I isolate the genomic DNA from Alzheimer's mice tails. I have done many PCRs, but this batch of isolated genomic DNA, has good concentration and...

08 September 2013 9,497 49 View

Can I use a combination of acids like Hcl and H2so4 / Hno3 in adjusting the pH of Tris buffer saline?

Tris buffer saline of pH-7.4. As in case of emergency like one of the above mentioned acid gets over while adjusting the pH, than rest of the pH could be adjusted using other alternative acid. Is...

06 July 2013 7,655 2 View

How to maintain a cell line N2a?

I see different people using different ways to maintain the cell culture. Can anyone please explain the basic steps involved in cell culture maintain, passaging etc. As to how much cells should be...

06 July 2013 516 2 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

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