PCR WOES...... - STRATEGY DOUBT... (Sep/28/2008 )
hello fellow molecular biologists,
I have a strategy doubt...can you please help me?
I have human genomic DNA, and primers for the promoter of a gene i wish to examine.
I will perform PCR of the human genomic DNA using these primers....so my promoter gets amplified.
Then using a restriction digest, i wish to cut out the promoter region from the amplified DNA..and carry out reporter construct studies....
Do you all think this is a foolproof strategy?
Can anyone point out any loopholes, if any; and how to get over them?
I am waiting urgently for your replies..please tell me.
best Wishes
megacool.
I have a strategy doubt...can you please help me?
I have human genomic DNA, and primers for the promoter of a gene i wish to examine.
I will perform PCR of the human genomic DNA using these primers....so my promoter gets amplified.
Then using a restriction digest, i wish to cut out the promoter region from the amplified DNA..and carry out reporter construct studies....
Do you all think this is a foolproof strategy?
Can anyone point out any loopholes, if any; and how to get over them?
I am waiting urgently for your replies..please tell me.
best Wishes
megacool.
Anything can go wrong at anytime anywhere. Essentially loopholes are going to be everywhere. Getting over them? This forum will do that or you could read papers for their methodologies or molecular cloning books.
PCR might not work, the reporter might act goofy( this is the part i hate the most), ligation not working? Who knows ... Just gotta prepare for the worst so that when they really do happen you won't go nuts!
TROUBLESHOOT is the key most of the time.
Have fun
CHEERS!!
I've been trying from time to time to make a reporter gene system with lacZ. I tried several transformations and I never get colonies. I just gave up. The plasmid construct was tested by my boss (I don't know how) and seems to be fine. So I have no clue why that particular transformation is so hard.
My boss said once that "cells are not deceptive, they don't like. Results are results" but in my very short experience, I can tell that no matter how close and carefully you follow the protocol, sometimes things don't seem to work the way they are supposed to and that really get on my nerves.
yeap been there done that. i designed such a nice cloning strategy , everything was going smoothly , checked everything on gel and restriction enzyme then lastly when testing the performance of the biosensor... it's not really a nightmare but a let down.
high background was my problem last time thus killing the fold-increase severely. If only i have more time i would have done some flanking with rrn terminator or something like that. . . . WHY DIDN"T I THINK OF THAT back then sigh...
p/s - mine was luminescence based ( luxCDABE) I make Agrobacterium shines~~~ tralala ( not literally, you can't really see them brighten up by ur own eye)