Cloning RACE products - Problem with sequencing ends (Oct/19/2004 )

Hello everyone.

I'm cloning 3' and 5' RACE products with TOPO system (Invitrogen).
All the protocol is OK (PCR amplification, cloning PCR products, screenning of transformants) but I have a problem with sequencing (with SP6 and T7 primers): I have only internal sequences with one of the primer but none with the other primer (corresponding with the ends of the cloned product).

Is it possible that I have a mixture of plasmids (inserts with different ends) in the same bacteria colony?

What can I do?

Thanks

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Hi

If your sequence gets unreadable at the vector/race product (exactly at the universal race primer/race product) border it is possible. In that case it is possible that you cloned different 5´- extension products. They might differ only in a few nucleotides. You can test this by sequencing from the other direction.
But it seems unlikely to me that you have several extension variants in one bacterial colony. Did you accomplish some single colony seperating steps?

greets

morrison

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Hi Morrison

If you use BDs SMART RACE there is an T7 primer in the adaptor and in the universal primer, so you can not use T7. Use another, there are often more than one sequencing primers in the vector.

Regards Caro

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Thaks Morrison and Caro

I think that, as Caro said, the problem is with the T7 sequence because this is in the vector (TOPO TA) and in the adaptor (BD SMART RACE).

I'll try with the M13 primer, and I'll tell you the results.

Best regards Matias

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