Hi,
I am designing gene specific primers for 5' RACE PCR. I am using the protocol in the attachment. I can see that the GSP-RT primer needs to be antisense regarding the mRNA sequence, but do GSP1 and GSP2 need to be sense or antisense?
Thank you!
Tatiana Kapelinskaya
Dear Britt, you're right, the primer should be antisense (revers complement) regarding the mRNA sequence and GSP1 and GSP2 need to be in the same orientation.They are used to reduce the occurrence of non-specific PCR products. I advise you to read the manual of the company Clontech.
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Britt Merlaen
so GSP1 and GSP2 also need to be antisense regarding the mRNA sequence?
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Tatiana Kapelinskaya
Of course yes! Try to paint the letters (ATGC) on both chains and will understand all . Don't forget to mark on the each of chain 5' and 3' ends.
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