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 3,056 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 3,012 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 6,266 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 696 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 2,257 16 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 9,577 49 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 9,644 5 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 7,749 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 629 2 View

How to confirm the site-directed mutagenesis result without performing NGS?

I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using...

08 August 2024 4,645 2 View

I can't see the ssDNA band after performing asymmetric PCR. Is there any way to do this?

After performing symmetric PCR, PCR purification was performed. Afterwards, asymmetric PCR was performed using the PCR purification product as a template, but no ssDNA band was confirmed in the...

08 August 2024 1,668 3 View

How much total RNA concentration to be extracted from sorted plasma cells from bone marrow of C57BL/6 mice for RT-PCR ?

i have sorted anti-NP specific plasma cells from bone marrow of C57BL/6 mice at certain times after immunization with variable counts and isolated total RNA using TRIZOL method for RT-PCR using...

05 August 2024 8,835 1 View

Could someone explain the basics of working with enzymes?

I'm interested in learning about enzyme usage. Could someone explain the basics of working with enzymes? I understand they're different from standard chemicals, but I'm uncertain about the...

01 August 2024 5,460 5 View

Does anyone have issues using Prepman Ultra reagent for MicroSeq ID bacterial, fungal and yeast sample preparation?

I have been attempting to extract DNA from Bacterial, Fungal and Yeast banked samples (>1e7 cells) using Prepman Ultra reagent and I seem to be struggling to obtain a sequence. Although the...

01 August 2024 2,079 0 View

PCR showing no bands - Master mix and primers didn't mix?

Hello everyone, I performed a PCR yesterday, and the results showed no bands on the gel. Of course, I probably missed some crucial steps, like adding my samples to the PCR strips themselves, for...

31 July 2024 2,406 6 View

Dimensions of an MJ Research 96-well alpha module?

Can anyone with an MJ research / BioRad PCR machine from ~2010 or earlier tell me the external measurements (LxWxH) of the removable standard PCR alpha modules that can be removed from a PTC200 or...

30 July 2024 2,867 0 View

What are some diseases that are caused by overactivity of enzymes?

I want to choose a resarch topic regarding enzyme inhibition. So I did my research and found out most of the diseases that originate from enzymes were actually caused by the "deficiency" of...

30 July 2024 2,483 5 View

How to retain amplicon yield after Ampure bead cleanup, following a 2-stage PCR protocol?

Hello all, I have been trying to follow a 2-stage PCR protocol used to amplify barcodes of a large yeast library, as per Nyugen et al. (2022) -...

30 July 2024 841 2 View

Someone have the key or the installer of the program Mx pro 3000 (PCR real time)?

I need to install this program

25 July 2024 4,756 0 View