I didn't design my primers that produce A overhang. And I have to do cloning. I read that I can use dATP and nonproofreading Taq to produce A overhang. Can I use this method to add an A to my PCR Product. I assume that My PCR Product is not blunt ended. Coukd you please help me about the isue?
If you have a PCR product generated using Platinum Taq DNA polymerase and you need to perform TOPO TA cloning, which typically requires an A overhang on the PCR product, you can indeed use a method to add the required A overhang to your PCR product.
Platinum Taq DNA polymerase lacks the terminal transferase activity necessary to add the A overhang, which is why your PCR product is not blunt-ended. To add the A overhang, you can use a non-proofreading DNA polymerase along with dATP in a PCR reaction. This approach is commonly referred to as "A-tailing."
Here's a suggested protocol for A-tailing your PCR product:
By performing A-tailing using a non-proofreading polymerase and dATP, you can add the necessary A overhang to your PCR product, enabling its compatibility with TOPO TA cloning. Remember to validate the presence of the A overhang in your PCR product by verifying its sequence or by performing a control ligation reaction without the insert to confirm successful cloning.
TOPO TA cloning kits are pretty efficient at blunt end ligation too. Even without an A overhang you can usually get some colonies. Easiest way to add the A overhangs is to just add 1 μl of the non-proofreading polymerase to your PCR product (no purification step) and extend for an additional 15 mins.
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