Screening of TA cloning by T7/Sp6 PCR possible? - (Apr/06/2005 )
Hi, all:
Our lab constantly uses Promega's pGEMT vector kit for TA cloning. Am just wondering if it's possible to use this vector's default T7 and SP6 to PCR and screen for the presence of my inserts, since the Tm of SP6 (29C) is extremely low? Thanks.
-bhchen66-
QUOTE (bhchen66 @ Apr 6 2005, 03:56 PM)
Hi, all:
Our lab constantly uses Promega's pGEMT vector kit for TA cloning. Am just wondering if it's possible to use this vector's default T7 and SP6 to PCR and screen for the presence of my inserts, since the Tm of SP6 (29C) is extremely low? Thanks.
Our lab constantly uses Promega's pGEMT vector kit for TA cloning. Am just wondering if it's possible to use this vector's default T7 and SP6 to PCR and screen for the presence of my inserts, since the Tm of SP6 (29C) is extremely low? Thanks.
i havent used the sp6 primer in colony pcr, but since the primer is used for sequencing (~40-50° annealing) it will also work in colony pcr. what works for sure are the standard uni (cccagtcacgacgttg) and rev (cacaggaaacagctatg) primers which will also bind to your pgemt vector. atb.
-zybrg-
oh and before i forget it, you just need to do blue/white selection to screen for clones with inserts. saves quite a bit of time....:-) cheers.
-zybrg-