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i_George's Content

There have been 5 items by i_George (Search limited from 20-September 18)


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#114339 DNA probe labeling for EMSA; How?

Posted by i_George on 05 July 2011 - 11:59 PM in Molecular Biology

Dear friends,

I try to do EMSA to check if my transcription factor of interest can bind with the promoter of my gene.
For the transcription factor, I express it in E.coli as a GST-fusion protein.

I have cloned the promoter of my gene and get some potential binding elements for the transcription factor.
Now, I plan to re-amplify the promoter in small fragments (180-300 bp)to use them as probes in EMSA.
In my lab, we used to use Biotin 3' end DNA labeling kit from PIERCE for single stranded-oligonucleotide probe.
In my case, they are double stranded and long probes and I guess this kit might not work ????

So I need you advice.
Do you guys have some kits that work well with my case?
or some other ways that can help?
(We cannot use radioactive labeling in my lab)

I hope I can get advice from you. Please help!!!



#101638 Looking for Pfu Taq for high GC-content template

Posted by i_George on 23 February 2011 - 05:21 AM in Molecular Cloning

If you mean CES from:
http://www.protocol-...lates-3469.html
I would say I haven't tried it. I just tried DMSO since my betaine have yet to arrive.

The product I use: 71975 KOD Xtreme™ Hot Start DNA Polymerase
http://www.merck-che...74AAAEj2Bl9.zLX
It does have it's own propriety ingredients which make it work.

Here's betaine solution from sigma-aldrich (which I bought): B0300-1VL
http://www.sigmaaldr...RAND_KEY&F=SPEC
Have not tried it yet... my work was done so no worries.


Here, in my lab we don't have betaine either.
I'm thinking that I should order betaine or a new DNA pol that can work well.
Which way is better you think?


Could you give me some reasons for using KOD Xtreme Hot Start DNA Pol. in stead of any other commercial ones?
So that I can tell my advisor about it efficiency and use it too
:)



#101613 Looking for Pfu Taq for high GC-content template

Posted by i_George on 22 February 2011 - 10:54 PM in Molecular Cloning

My recent experience:
I was trying to amplify 2 highly GC rich genes for cloning, tried DMSO not working. Ordered betaine solution but not yet arrive. So I had asked for a sample of KOD extreme hot-start polymerase (with proof reading). Although there are many unspecific bands and smearing, but at least I still able to gel excise my desired band. Now it turns out to be the sequencing machine problem which can't sequence one of it.


Have you ever tried additive CES ?
I don't know if it works?



#101612 Looking for Pfu Taq for high GC-content template

Posted by i_George on 22 February 2011 - 10:39 PM in Molecular Cloning

I would start by adding 5% of a 1 M Betaine solution.


phage434--thanks for your reply
I will follow your advice
However,would you tell me what kind of betaine you use?
You mean Glycine bataine/trimethylglycerine, right?



#101567 Looking for Pfu Taq for high GC-content template

Posted by i_George on 22 February 2011 - 10:46 AM in Molecular Cloning

Hi there,
I'm a new in molecular lab and new member in this board too, so pls help me..
I'm dealing with PCR cloning of templates with high GC-content (70.9, 67.3, 63.9, and 52.3% respectively)
I want my PCR product to be blunt-ended and proof-read
So far, I used Pfx50 DNA pol,invitrogen but it did not work at all

Some people talk about adding some additives, but I don't know where to start?
or do you guys have some Taq to recommend?

I'm looking forward to your responses




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