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Proofreading polymerase problem


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#1 lemmiwinks

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Posted 30 January 2009 - 01:40 PM

Hi guys,
I'm amplifying genomic DNA to get a product ~2.5kb in length but using Taq polymerase incurs a few base pair additions/deletions/modifications which aren't helpful with future cloning. So I used Roche Expand High Fidelity plus polymerase with proofreading capability which resulted in no band whatsoever (after 1 month of tweaking everything, with very little effect). So i changed to Finnzymes Phusion which resulted in a band straightaway! However, i now have a problem of inserting blunt end PCR products into a cloning vector. Have tried pBlueScript cut with EcoRV and SmaI, dephosphorylated and transformed into DH5alpha with no luck. I have also tried TA cloning and ligating this into pGEM-T but again I have had no luck. I've tried digesting the PCR products to create sticky ends, but I don't think there are enough nucleotides on the end to achieve this. I'm at a total standstill. Any help would be greatly appreciated.

Lemmiwinks

#2 perneseblue

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    Unlimited ligation works!

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Posted 30 January 2009 - 02:24 PM

since you PCR amplified your DNA fragment with phusion, you have to treat it with taq to add the A overhangs, for TA cloning.

what conditions have you used to conduct the blunt end cloning?
Try lowering the ligation temperature (16C or 4C) and increasing the ligation time (overnight). Increasing the concentration of insert and vector in the ligation mix does help too. Adding PEG6000 to a final concentration of 10% to the ligation mix (or using quick ligation buffer.. which has PEG6000 pre added) does help blunt end ligation.

As for sticky end cloning... have you looked at NEB to make certain that it isn't possible? Some restriction sites such as EcoRI and BamHI require few bp for efficient digest. Even having one end sticky and the other end a blunt end cloning would help.
May your PCR products be long, your protocols short and your boss on holiday

#3 Nathan

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Posted 31 January 2009 - 02:29 AM

hey try TA cloning.. it should work.. after ur regular PCR, incubate the reaction mix with 1 unit of Taq pol for 15 mins.. this time should be sufficient for adding A overhangs. then extract PCR product by phenol-chloroform sodium acetate 100% ethanol and air dry it.. then suspend pellet in TE buffer.. i guess this should work.. alternatively u may use a Zero Blunt PCR Cloning Kit from introgen.. guess this may help.. :lol:

#4 Julio-Claudian

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Posted 31 January 2009 - 05:24 AM

perneseblue and nathan,

When you guys mentioned TA and treating with Taq, is it at the elongation temperature of 72'C? If yes, then the DNA strands didn't separate/unzip; does that mean Taq can add bases in adjacent manner instead of the usual complementary addition? I'm confused.

Do we do TA clone using a kit or just a simply addition of Taq to the blunt end products?

#5 perneseblue

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Posted 31 January 2009 - 06:42 AM

perneseblue and nathan,

When you guys mentioned TA and treating with Taq, is it at the elongation temperature of 72'C? If yes, then the DNA strands didn't separate/unzip; does that mean Taq can add bases in adjacent manner instead of the usual complementary addition? I'm confused.

Do we do TA clone using a kit or just a simply addition of Taq to the blunt end products?


Yes, we do mean elongation temperature at 72 C with dATP. And yes it does. This is a quirky characteristic of Taq polymerase when in the presence of dATP and blunt end DNA molecules for a prolong period of time (~10 - 20mins). Although Taq only adds this extra A overhang to 70% of the molecules.

The TA vector has a 3' T overhang. And the insert must be made to have a 3'A overhang. Some "DNA polymerase" sold by companies are actually a mixture of Taq and a proofreading polymerase (usually pfu) in a ratio of 10:1 to 30:1. These mixtures result in the PCR products to have an 3' A overhang, that can be used for TA cloning.

Your TA cloning kit only contains the vector with a 3'T overhang, and components for the ligation reaction. You need to provide the PCR product with an 3' A overhang. So once you have used Taq to add 3' A overhang, clean up the DNA insert. Then add the DNA to the TA cloning kit to ligate it into the TA vector.
May your PCR products be long, your protocols short and your boss on holiday

#6 Julio-Claudian

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Posted 01 February 2009 - 10:19 PM

Fantastic. Thanks a lot!




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