Adding 3' A-overhangs for TA cloning - (Jul/14/2008 )
Hi, I'm using Phusion for PCR and then cloning it into TA cloning kit from invitrogen.
According to the TA cloning kit, I have to add 3' A-overhangs, and they provided this protocol:
1. no need to change buffer, just add Taq
2. incubate 72C 8-10mins
3. Extract immediately with phenol-chloroform
4. Add 3M Na-Acetate and 100% Ethanol
5. Centrifuge to precipitate the DNA
6. Wash with 80% EtOH
7. air dry
8. Reconstitute with TE by the starting volume of reaction
It that NECESSARY??
is there a reason not to using the product after step 2 to do TA cloning? Because if I am using Hi-Fi Taq instead of Phusion, there is no need to purify the PCR product to get rid of the buffer, I do not totally understand why steps 3-8 are needed
Can anyone explain to me why?
I guess it's because the Phusion has the proofreading-exonuclease activity that will remove your overhangs in abscence of dNTPs...It's the best explanation I can come up with...
But in my opinion, you could use the product after step 2. directly if you would use a TOPO-TA cloning kit, because the TOPO reaction is so fast... (I've never tried that though...)
With the phenol extraction you will lose lots of sample, try to use a spin colunm purification (no gel extraction, because you will lose the Poly A again).
The TOPO TA cloning instruction manual I have has you go directly from the Taq to the ligation without purification. It says you can purify by gel if you have multiple PCR bands. Now there is one line in smaller type that says if you plan to store your sample overnight before proceeding with TOPO cloning, extract your sample with an equal volume of phenol-chloroform to remove the polymerases. Ethanol-precipitate the DNA and resuspend in TE buffer using the starting volume of the PCR. Seems like this is the most difficult method though, since you can get rid of the polymerase using a PCR purification column.
I used to cloned with TOPO TA and as told by rkay I didn't purified the PCR. Just made the PCR, stored at -20C and during the week I cloned following the kit protocol. Never had a problem with it.