Suraj Kumar Mandal

11 Questions 21 Answers 0 Followers

Questions related from Suraj Kumar Mandal

How to avoid protein precipitaion ?

I am working on a thermophilic protein (pI: 7.72) purified using Ni-NTA chromatography (Final buffer: 25mM Pipes (6.6), 300mM NaCl, 1mM PMSF, 10% glycerol, 200mM imidazole). But the protein...

10 October 2016 8,316 24 View

Does cloning vectors (pET series specially) confers toxicity to the gene/protein of interest?

I am working on a thermophilic protein which is not known to be toxic. It was earlier cloned in pET11a and cell death was observed after 2-3 hrs of IPTG (0.1mM) induction. I then cloned the gene...

07 July 2015 795 4 View

Why do I detect two proteins after purification?

While eluting two of my proteins with 250mM of Imidazole, I am getting two different bands (Size of the proteins are mentioned in the Image attached). The genes are cloned in pET22b without Signal...

07 July 2015 1,760 8 View

Is it recommended to incubate Thermophilic genes at 42 degree celsius after IPTG induction?

In some papers people have recommended to use incubation at 42 degree celsius for short time for protein expression after IPTG induction? If YES, What will be its effect on protein's...

12 December 2014 3,469 5 View

Why are my non-specific bands coming even after PCR clean up?

I am trying to clone a T.thermophilus gene. I have used 3%/5% DMSO during PCR amplification (using phusion enzyme). Amplification is coming but after PCR purification, and I am also getting...

06 June 2014 2,224 21 View

Why is the ligation of a gene with different vectors not coming?

I have tried cloning of my gene of interest in pET22b and pET28a, for different ratio, but the ligation didn't work (though colonies are coming in some cases). I am pretty much sure that my PCR...

05 May 2014 6,649 14 View

Do you have any suggestions on how to avoid primer dimers after PCR clean up?

When I am doing PCR clean up after PCR amplification (using phusion + DMSO) of a thermophilus gene, and along with the amplified gene, the primer dimer is also appearing. Do you have any...

04 April 2014 1,441 18 View

Is there any protein expression host designed for thermophilic bacteria? Can it be expressed in E.coli hosts namely Rosetta, C43 DE3 or not?

I am trying to purify a protein from a thermophilic bacteria (gene cloned in DH5 alpha) but I am not getting expression of my protein after IPTG induction. I have tried with different expression...

03 March 2014 3,015 11 View

What is the denaturation temperature used for plasmid vectors (circularised) in PCR? Is it different from 95 degrees C?

I am amplifying a gene from pET11a vector. Is the temperature for denaturation (95 C for chromosomal DNA generally) different for amplifying the gene from plasmid DNA?

03 March 2014 6,305 5 View

Which part of the alsA gene in E.coli is bound to the transmembrane domain?

alsA gene has two sub-units and some part of one sub-unit is bound to the TMD (alsC).

11 November 2013 9,682 0 View

Is alkaline picrate method considered a less sensitive method for creatinine estimation?

What are the other substances which can be used that give a positive result with alkaline picrate?

11 November 2013 8,895 3 View