direct digestion of colony PCR product

Zhonglin Chai via methods%40net.bio.net (by Zhonglin.Chai from bakeridi.edu.au)
Thu Apr 7 18:57:42 EST 2011


You may find it essential to purify your PCR products before restriction enzyme digestion if you want to clone the PCR products. Without purification, the sticky ends of the DNA generated by the restriction enzyme(s) will be destroyed in the same time in the same reaction due to the presence of the DNA polymerase activity and/or proof reading activity of the enzyme (Taq, pfu, ect) you have used for your PCR. 

Zhonglin Chai

-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Dunowska, Magda
Sent: Friday, 8 April 2011 7:32 AM
To: Marta; methods from magpie.bio.indiana.edu
Subject: RE: direct digestion of colony PCR product

Marta, I think that as long as your restriction enzymes are compatible with your PCR buffer you shouldn't have any problems digesting the PCR product without prior purification. If unsure, just try it - it's not a lot of time/effort to add the restriction enzyme to the un-purified PCR product, incubate it for the desired length of time, and then run the digest on a gel...you'd have to cut the bands out of the gel for ligations.
Good luck,
Magda

-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Marta
Sent: Friday, 8 April 2011 1:38 a.m.
To: methods from magpie.bio.indiana.edu
Subject: direct digestion of colony PCR product

Dears,
I made a colony PCR to verify my clones in that way: I picked up a colony to PCR mix: Buffer (10X) 3.0µL, DNTPs (2.5 mM each) 3.0µL, Taq
(5 units/µL), Forward Primer (20 µM) Reverse Primer (20 µM) Water; TOTAL 30µL.
Now I want to digest my PCR product by: BamHI an XhoI and the ligate insert to expression vector pET28b+.
To save time and money I want to digest PCR mixture directly without any purification. I am not really sure that this way is correct. Do contaminations like bacteria cell components disturb restriction reaction?
I know that they can disturb.... but I don't need 100% efficiency.
So is there a posibility to obtain positive digestion and then ligation?

Please help me
Marta



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