any good method for rapid screening for recombinant plasmids? - (Aug/30/2006 )
These days, I was working on cloning 7 different genes into TOPO pCR2.1 vector (Invitrogen) and found it extremely time-consuming and expensive to perform tons of colony-PCR. So I was wondering if you guys have any good method for a rapid, relatively cheap screening prototol to detect the recombinant?
Another question to ask: did anyone ever use Platinum Taq High Fidelity polymerase for amplifcation of insert and then ligate into TOPO vector? Is A-tailing addition treatment required? What is the efficiency? What I did was to amplify the insert using that enzyme, gel purify and add A-tail and then ligate. Although I got both blue and white colonies showing up in LB+Kan50 plates ,the majority of white colonies picked for colony PCR screening was negative. So it seemed to me that the ligation efficiency was not that great even though I added A-tail. Any good ideas to improve that?
thanks for help!
Since you're working on single gene for each ligation and transformation, you don't really have to screen very large number of colonies. Less than 10 would be suffice, though it's still time consuming.
Regarding T/A cloning, could refer to http://www.protocol-online.org/prot/Detailed/3827.html.
Could try adding more insert into the insert:vector ratio. I'd normally add as much insert DNA as possible.
thanks---the reason I was performing lots of colony-PCR --I found the rate of having positive amplification signal was rather low (about 25%). So I was hoping to increase the number of PCR running to find more recombinant. But that burned bucks and was extremely time-consuming....
Colony PCR is one of the simplest and least painful screening techniques. Perhaps you are working too hard when you set them up. 10 ul reactions are sufficient, a master mix with primers can be used to set up the reaction, and you need not do anything to the cells except to dilute a colony into 50 ul of water and add 0.5 ul of the result. You spot another sample of the water onto a master plate, and you are done. The lysis of cells happens in the 5 minute 95C initial heat of the PCR cycler. With 10 ul reactions, it is also quite inexpensive.
yeah, you are right--I at first used 25ul reaction for each colony--so that cost lots of reagents to setup.
today, I setup a 10ul-reaction and followed your protocol and it worked extremely well! thanks!
I dip little bit bacterium (as little as possible)at the bottom of PCR tube and then add 10 ul PCR reaction mix for PCR (25 cylces).