In a gateway cloning system, we attach CACC to forward primers to achieve site specific recombination between our templates and vectors. What happens when the CACC sequence is present along with one end in the fragment of interest?
Hello Bhupendra,
you don't need to worry when you have a CACC in your gene of interest. The CACC at the 5' end of the primer is a single-stranded overhang that pairs with the overhang sequence, GTGG, in the pENTR vector. The CACC in your gene is double-stranded so no pairing is possible.
With best regards!
thanks Scheckhuber,
but after PCR amplification how would you get overhang region, as we know after 3rd cycle of amplification all overhang region get double stranded.
As i know gateway system is recombination based system not ligation therefore there is a possibility of getting truncated insert.
You are right. I checked the Gateway manual and it says: "The overhang in the cloning vector (GTGG) invades the 5′ end of the PCR product, anneals to the added bases, and stabilizes the PCR product in the correct orientation."
This invasion is probably a lot easier (and therefore much more likely) at the end of the PCR product than in interior regions. This could explain why you extremely rarely get truncated inserts.
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