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degenerate PCR


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#1 sowmi

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Posted 21 May 2010 - 01:49 AM

Hi all,

Can someone help me out with cloning of degenerate PCR products into a T/A cloning vector. The chosen vector is pGEMTeasy and I would transform it into DH5 alpha cells followed by blue/ white selection and then sequencing. The PCR enzyme suggested was BD advantage (from clontech).

1. Does this enzyme produce T overhanga?
2. Is this enzyme have enough proof reading activity to enable proper sequencing of the gene?
3. Is the product formed compatible to be cloned into the respective vector?
4. Is pGEMTeasy cloned and selected properly with DH5alpha?

thanks to all members.

#2 perneseblue

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Posted 21 May 2010 - 07:50 PM

Could you post a link to the enzyme that you are using. The closest thing on Clontech's website is Advantage HD Polymerase Mix. I was unable to locate BD advantage.

Advantage HD Polymerase mix is a proof reading enyzme mix and thus unsuitable for direct TA cloning. You could use a treatment with Taq polymerase to add the A overhang to the PCR product. Just add your PCR product to Taq and dATP and heat at 72C for about 30 min. That would add the A overhang.

pGEMTeasy should be okay in DH5alpha.

Hi all,

Can someone help me out with cloning of degenerate PCR products into a T/A cloning vector. The chosen vector is pGEMTeasy and I would transform it into DH5 alpha cells followed by blue/ white selection and then sequencing. The PCR enzyme suggested was BD advantage (from clontech).

1. Does this enzyme produce T overhanga?
2. Is this enzyme have enough proof reading activity to enable proper sequencing of the gene?
3. Is the product formed compatible to be cloned into the respective vector?
4. Is pGEMTeasy cloned and selected properly with DH5alpha?

thanks to all members.


May your PCR products be long, your protocols short and your boss on holiday

#3 sowmi

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Posted 23 May 2010 - 08:38 PM

Thanks for your reply...
I am attaching the BD advantage enzyme user manual with this msg..

Attached Files



#4 perneseblue

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Posted 29 May 2010 - 08:20 PM

The answer appears to be yes. Titanium Taq from BD bioscience clontech can be used in TA cloning.
May your PCR products be long, your protocols short and your boss on holiday




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