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PCR with overlapping primers - (Apr/04/2007 )

hi,
I want to amplify upstream and downstream flanking sequences of a gene and clone the two fragments into a vector. the two inside primers are overlapping ones. I am thinking the correct way is to amplify the two fragments seperately and then mix them and do the PCR again to connect both with overlapping segments. Can any one help me out with exactly how to do this???

-nair-

QUOTE (nair @ Apr 4 2007, 09:25 PM)
hi,
I want to amplify upstream and downstream flanking sequences of a gene and clone the two fragments into a vector. the two inside primers are overlapping ones. I am thinking the correct way is to amplify the two fragments seperately and then mix them and do the PCR again to connect both with overlapping segments. Can any one help me out with exactly how to do this???


I don't understand why need to amplify separately while you still need both genes joined before clone them.

Although you didn't elaborate I'll assume you want to introduce that overlapping sequences, right?

If so you're correct!! Amplify the two genes separately then assemble new PCR rxn mixture and add small amount of you previous PCR products in one tube and allow joining for about 10 to 15 cycles under appropiate PCR condition.

Dilute the joined product ~25x in the new pull-through PCR and use primers that anneal at the ends of the newly constructed gene for amplification.

If possible, can you enlighten me on the main goal you want to achieve here? because sometime multiple amplification of one gene result in mutation either base substitution or deletion.

-chick gene-