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Questions related from Gaurav Kumar
There's a clone in pBAD18cm vector (6800bp) which was subjected to point mutation through PCR-based technique. After PCR and DpnI digestion an extra band of approx 1500bp was observed in cloned...
02 February 2016 4,658 4 View
I am doing enzyme kinetics studies. I need to derive certain parameters for a representable data. The present MS excel sheet fails to provide enzyme kinetics parameters such as Km, Kcat, Vmax...
11 November 2015 7,379 7 View
A very basic query regarding the solubility, stability and storage of carbapenems antibiotics such as Meropenem trihydrate or Imipenem monohydrate. I have known to dissolve them in DMSO in higher...
10 October 2015 5,507 8 View
Here I have attached SDS-PAGE gel picture of Ni-NTA affinity chromatography elution profile of a protein at varying concentration of imidazole in tris buffer(pH 8). I have done washing in 10, 25...
08 August 2015 1,795 8 View
It's confusing to find extra bands of higher molecular weight in my insert sample which was PCR purified by gel extraction method. The insert bands were very clean in 0.8% agarose gel. It was...
06 June 2015 4,371 13 View
What are the good servers for detecting signal peptides apart from SignalP 4.1 and Signal blast? Sometimes these servers fail to detect the presence of signal peptide in a given protein sequence....
05 May 2015 8,528 2 View
I tried to check the presence of signal sequence of full length construct (amino acid) of NDM-1 using SignalP 4.1 server but could not find it. Can any body suggest anything in this matter? Thanks
05 May 2015 8,502 0 View
I am checking in-vivo expression of a protein(full length) in pBAD18cm vector using BL21 expression host. I am getting an extra band of protein, 10kDa higher than expected in SDS-PAGE as compared...
05 May 2015 6,611 10 View
I have cloned a beta-lactamase gene in BL21 E.coli strain using pET28a vector for expression and in vitro studies. After Ni-NTA column purification of this His Tagged protein, I am getting two...
01 January 2015 2,435 16 View
My His-tagged purified protein contains 10mM trisCl, 300mM NaCl and 200mM imidazole at which the protein was eluted at pH 8.0. What should be ideal dialysis buffer composition to remove...
01 January 2015 5,964 10 View
I want to make my gene construct as soluble devoid of signal peptide and helical amphipathy to avoid membrane anchor. Can anyone suggest few examples. Thanks.
12 December 2014 7,103 2 View
This is quite amazing. I used old TAE buffer to run 0.8% agarose gel at 80 volt electric potential. I found unusual DNA band from vacant wells. When I replaced the old buffer with fresh buffer the...
11 November 2014 5,914 7 View
I am studying in vivo expression using pBAD18 Cm vector in BL21 cell strain. I am confused about the induction procedure. I am using 0.2% arabinose to induce pBAD promoter at 0.2 OD growth. Can I...
11 November 2014 7,162 9 View
Can we run the SDS PAGE resolving gel at the same pH as running buffer pH or vice versa? Any logical explanation?
11 November 2014 1,310 7 View
I am amazed with human nature. Sometimes I feel we have two brains or at least mirror image of single brain with opposite nature. One part is full of positive energy, positive hope, kindness,...
11 November 2014 3,375 0 View
I set up a PCR reaction volume of 50ul with Taq polymerase and I found distinct bands of amplicon but when I double the reaction volume to 100ul to get higher volume of amplicon I found multiple...
11 November 2014 6,731 17 View
I have observed recently. My protein forms a solid mass after heat denaturing with loading dye. It gets precipitated!! Why is it so?
09 September 2014 7,504 5 View
I performed SDS-PAGE gel assay and found that only four protein marker bands are visible instead of seven! I am confused about the size of marker bands which have disappeared. I have repeated the...
09 September 2014 8,874 9 View
I applied 140, 120. and 100 volts and I have obseved a very random migration of protein sample. It may be due to presence of salts (imidazole, NaCl, TisCl) in the sample. Sometimes the sample gets...
09 September 2014 8,175 5 View
I have loaded protein sample eluted from Ni-NTA column with different concentration of imidazole. I have found that the migration of of protein sample in 12% SDS-PAGE is not smooth or in a...
09 September 2014 9,794 22 View
Protein foaming during sonication is a sign of protein degradation. I tried my best to avoid it by using low amplitude (up to 60%) sonication but still i find foaming in the protein sample which...
09 September 2014 8,985 4 View
Just for curiosity. Do cancer stem cells (CSCs) really exist or they are just derived from normal tissues. Can their origin be traced?
09 September 2014 3,957 11 View
I am having a problem with conversion of enzymes concentration. Enzyme concentration is given in Units/mL but I want to convert in weight/volume. Please provide your suggestions.
08 August 2014 7,570 17 View
I am trying to purify membrane bound beta-lactamase protein in BL21 E.coli strain. I have created soluble construct of the gene by removing the signal sequence from 5' and 45 nucleotides removed...
08 August 2014 8,266 8 View
I prepared 12% SDS-PAGE gel using sambrook manual and found that the gel expands after keeping it in staining solution for few hours. What could be the probable reason? I checked the running...
07 July 2014 9,572 7 View
My plasmid construct has two shine-dalgarno sequence. One is present 55bp upstream of gene and other is present in my primer. Does the presence of two SD affect the expression of my gene?
07 July 2014 1,958 5 View
I want to do knock out of SHV class of beta-lactamase from Klebsiella pneumoniae genome as a part of my PhD work. I am confused with the techniques available. I have never done knockout before....
07 July 2014 5,135 2 View
I have read about the fragile nature of transformed BL21 cells for expression studies. I have been using the transformed BL21 cells stored at normal refrigerator temperature for expression...
07 July 2014 7,232 6 View
I have cloned my gene in pET28a vector within XbaI and SacI site. I realised that SacI site has omitted His tag at the carboxyl terminal in pET28a vector due to frame shift. Can anybody suggest...
03 March 2014 5,132 3 View
blaLEN and bla SHV are a class of beta lactamases that has resulted in evolution of drug resistance in Klebsiella world wide.
02 February 2013 9,396 0 View
Interesting finding. I am eager to know, how did you isolated and sequence such a large plasmid?
01 January 1970 779 2 View
