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)
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?
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?