Gautam Krishnan

10 Questions 51 Answers 0 Followers

Questions related from Gautam Krishnan

Hello!I have a monocytic cell line THP-1 .Can I use it to do cytokine upregulation studies without adding PMA.?

I am trying to simulate an in vivo situation in host macrophages by adding a recombinant protein that is expressed in high amounts.Can i do cytokine upregulation studies without adding PMA ?Will...

12 December 2017 7,140 2 View

How to optimise media composition to improve expression of a clone ?

I have a clone that expresses in E.coli BL21DE3 and BL21DE3 star cells.Ive found that the protein band is only seen at higher temperatures and not at lower temperatures .Can media optimisation be...

05 May 2016 5,751 5 View

How to explain loss of expresion in BL21DE3 cells?

I have a clone of a protein in pET21a and have tried expressing it in BL21DE3 cells and recently BL21DE3 star .On both occasions I have found loss of expression. I see a protein band with freshly...

04 April 2016 5,453 7 View

How can I identify His tag protein when the level of induction is poor?

I have expressed a protein of around 18Kda with C terminal his tag in pET21A.I have very low level of expression in pellet with protein going in pellet.I did low temperature expression to get...

02 February 2016 5,217 3 View

Is the use of freshly transformed BL21DE3 recommended for each protein expression?

On this forum I have seen questions being asked on how to reproduce expression in BL21DE3 and have found the standard advice is to freshly transform BL21DE3 cells with plasmid each time for...

01 January 2015 9,336 10 View

Why is the mobility of uncut vector versus cut vector different? How do you explain higher mobility of uncut versus linearised?

I have done digestion of vector pET21A with EcoRI and am seeing higher mobility of uncut versus cut vector? What could be the reason? Could I still go ahead with the cloning if the linearised...

12 December 2014 4,514 10 View

How do I know that double digestion of Pet21 a with Bam and Xho 1 has proceeded to completion?

I am trying directional cloning into pET21 A ?How do I know that double digestion has worked?I am running a 0.6% gel to look for"invisible" uncut DNA.Is there are a better way to know?

08 August 2014 4,521 23 View

What modification do I need to do to make the ligation reaction work?

I tried ligation of pET 21 A and my insert 624 bp with the following conditions Tube 1 :Vector alone Tube 2:Vector +Insert 16 hrs at 16 degrees C for 13 hours Vector : 80 ng- in terms of copies...

03 March 2014 7,272 21 View

How do I ensure the right amount of ligase for Pet21 A cloning?

I tried a ligation of 524 bp fragment into pET21 A vector 0.022 pmols of Vector-80 ng versus 0.121 pmols of Insert -50 ng I used Ligase Weiss units 0.28 as per pet manual -On running the gel of...

03 March 2014 2,633 8 View

Does anyone have experience with pRSETA ligation with a 624 bp insert and getting positive result when doing PCR but no release of insert?

My reasoning is that the EcoRI site is lost after ligation Another issue is that the Self colony is also showing positive in PCR after ligation Please see gel pictures and suggest me a reason for this

12 December 2013 1,467 6 View