adding A overhangs - how much dATP? (Aug/10/2005 )

I need to add A overhangs to my purified blunt-ended PCR products with Taq polymerase. How much dATP should I add in this reaction? Thank you.

s70048

Hiya,

From the Promega pGEM-Teasy booklet:

Start with 1-7ul of purified PCR fragment
Add 1ul Taq Polumerase 10X reaction buffer with MgCl2
Add dATP to a final concentration of 0.2mM
Add 5U Taq DNA Polymerase
Add water to 10ul
Incubate at 70C for 15-30min
Use 1-2ul in a ligation reaction

Good luck!

Nicole

Hiya,

From the Promega pGEM-Teasy booklet:

Start with 1-7ul of purified PCR fragment
Add 1ul Taq Polumerase 10X reaction buffer with MgCl2
Add dATP to a final concentration of 0.2mM
Add 5U Taq DNA Polymerase
Add water to 10ul
Incubate at 70C for 15-30min
Use 1-2ul in a ligation reaction

Good luck!

Nicole

0.2mM is a bit of overkill- anything over 20 µM will be fine. Taq's Km for dATP is very high.

Daniel

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