Gaurav Kumar

31 Questions 99 Answers 0 Followers

Questions related from Gaurav Kumar

Extra DNA band in DpnI based point mutation?

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 4,644 4 View

What are the best free softwares to study and analyse enzyme kinetics data?

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 7,367 7 View

What is the best solvent for carbapenem antiobiotics? Water or DMSO?

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 5,493 8 View

Are there any ghost bands in my SDS-PAGE gel or contaminating proteins?

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 1,777 8 View

How do I get higher molecular weight band after PCR clean up followed by gel extraction of double digested inserts?

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 4,351 13 View

What are the good servers for detecting signal peptides apart from SignalP 4.1 and Signal blast?

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 8,515 2 View

Does NDM-1 have signal sequence?

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 8,488 0 View

Is it possible to get a protein of interest at higher molecular weight than expected in SDS-PAGE?

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 6,595 10 View

Why there are two bands in SDS-PAGE of a purified protein instead of one?

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 2,420 16 View

What should be the ideal dialysis buffer composition for a His-tagged purified protein to remove imidazole salts?

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 5,950 10 View

What are the good servers or websites to study protein structure, signal peptide sequences and helical amphipathy?

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 7,090 2 View

Can we get DNA bands without loading any sample?

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 5,903 7 View

What are the ideal conditions for inducing pBAD promoter?

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 7,148 9 View

How is operating pH in SDS PAGE resolving gel 9.5 if the running buffer pH is 8.3 and the gel pH is 8.8?

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 1,296 7 View

What is mental conflict? Does it really affect the course of human development?

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 3,361 0 View

Why am I getting multiple bands in PCR?

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 6,715 17 View

Why does a protein form a precipitate before loading in SDS-PAGE?

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 7,490 5 View

Can anyone help me find out why my protein marker bands disappear?

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 8,860 9 View

A very general query: What is the normal voltage condition for SDS-PAGE containing Ni-NTA eluted protein samples?

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 8,166 5 View

Why the protein moves Zig Zag way in the SDS-PAGE?

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 9,780 22 View

How to avoid protein foaming during sonication?

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 8,970 4 View

Do cancer stem cells really exist or are they just derived from normal tissues? Can their origin be traced?

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 3,937 11 View

How to convert Enzyme units into weight by volume unit?

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 7,552 17 View

Which method yields better result: lysis buffer or sonication or combination of both for membrane proteins?

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 8,251 8 View

Why does my SDS-PAGE gel swell after staining? It gets expanded and delicate!

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 9,556 7 View

Does the presence of two Shine-Dalgarno sequence affect the expression of my gene?

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 1,944 5 View

Beta-lactamase gene knockout in Klebsiella pneumoniae.

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 5,120 2 View

What is the shelf life of transformed BL21(DE3) E.coli cells in a normal refrigerator?

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 7,214 6 View

Can anybody suggest a solution for how to get my clone with His tag in frame using the same pET28a clone?

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 5,120 3 View

Has anybody worked on blaLEN and blaSHV beta lactamase genes? Can you suggest appropriate vectors to clone and express these genes?

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 9,373 0 View

How did such a large plasmid isolated and sequenced?

Interesting finding. I am eager to know, how did you isolated and sequence such a large plasmid?

01 January 1970 763 2 View