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Bpaul

Member Since 29 Jul 2012
Offline Last Active Jan 06 2013 08:42 PM

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bioforum
16 Sep 2012 - 19:23

Topics I've Started

GST-tagged protein expression problem! :-(

29 August 2012 - 08:49 PM

Hey All

I am trying to express a GST-tagged protein (gene cloned in pGEX-4T-2 vector!). My protein is around 24 kDa. I did check the reading frame and all that and its all good! I tried expressing the protein in BL21(DE3) at different temperatures (37, 30, 25 and 16 degC) with different IPTG concentrations (0.1 mM and 1mM). And now i see proper bands around 25 kDa (checked using SDS-PAGE and western blots!) and i believe its the GST! Whereas my required protein (25 kDa + GST) is no where to be found! Posted Image

I am thinking either the protein is not expressing or its expressing but the sonication must be making the GST to fall off?? I am yet to check the uninduced one! Any suggestions? Any ideas? Thank you very much!

BP

DNA sequencing after ligation! HELP!

26 August 2012 - 05:35 PM

Hi All

I am pretty new in molecular biology and hence this question!

So i got the ligation to work and then sent it for sequencing. I used the PCR primers (primers i used for the PCR reaction!) for sequencing! So my protein is 210 amino acids long. But i will use a random protein sequence to ask my actual question. So consider this is my protein sequence...

MDLDIKQSQL AATNRRHGKW DEWSDKRESR VWKTDCRIFG

I used both forward and reverse primers (as seperate reactions!) for sequencing. I was hoping to see the entire protein sequence in my DNA sequencing result! But instead i saw the results like this..

Reaction no.1. With Forward primer:

The resulting sequence showed match with my protein sequence from AATNRRHGKW DEWSDKRESR VWKTDCRIFG and the ones before (N-terminal ones!)that was something else!

Reaction no.2. Reverse Primer:

The results showed match with my protein sequence MDLDIKQSQL AATNRRHGKW DEWSDKRESR and the end portion was something else!

Both the reaction mixtures were set up from the same DNA tube! so when i look at the bigger picture i have my insert! But i was hoping that the results will be coming out as the entire insert and not like what i got! So my question is... is this normal? Is this how it should be?

Sorry about this very stupid question! But please help! Will be greatly appreciated! Thank you Posted Image

Clarification on digesting a ligation mixture!

01 August 2012 - 08:17 PM

Hi all (and badcell!)

I would like to requote badcell's post!

badcell, on 26 January 2005 - 10:41 AM, said:
In theory it's not absolutely necessary to dephosphorylate if you are using two REs with cohesive ends, but in fact, if the restriction efficiency of the enzymes is not 100%, you will get some molecules which have been cut only with one or the other enzyme, so they will religate if they are phosphorylated. One useful trick when cloning using two different sites is checking if you loose any site in the vector multiple cloning site (if any site is between the two REs you are using and not anywhere else in the vector). If this is the case, and this site is not found neither in your insert, you can digest your ligation with that enzyme and you will cut only your religated plasmids and not the ones with your insert, which will not have that site. So you will get only colonies with the insert.

This actually sounds great and i want to try it! But i do have a question... please correct me if am wrong! When we RE double digest the vector, we always gel purify it. So i am guessing every unwanted bit will be gone (including the middle bit between the two RE's we used!). So if the vector gets religated (during the ligation!), then there wont be a middle portion!! Right?? Posted Image

Can anyone please explain this to me?

Thanks in advance!

BP