Dear all,
Good day and greetings.
I'm just wondering if it's possible to perform PCR, once my cDNA fragment is inserted into Novagen's pET series vectors, using the above T7 pro/T7 term. as primers since their respective GC% and Tm are fairly low? Does any one of you have prior successful experience with this? Could you kindly provide your PCR settings as well? Many thanks in advance!
Hi Suliman,
You should have no problem using T7 forward and rev primers to amply your insert. I commonly use these primers for colony PCR to check for correct subcloning. I typically will cycle 95 degrees for 30 secs, 60 degrees for 30 seconds, and 72 degrees 30 seconds per 1KB, however these setting are somewhat polymerase specific. One thing to keep in mind though, is if you are using a vector that has GST or MBP, using the T7 forward primer will also amply the tag and your insert.
Good luck,
Joe
Hi Suliman,
You should have no problem using T7 forward and rev primers to amply your insert. I commonly use these primers for colony PCR to check for correct subcloning. I typically will cycle 95 degrees for 30 secs, 60 degrees for 30 seconds, and 72 degrees 30 seconds per 1KB, however these setting are somewhat polymerase specific. One thing to keep in mind though, is if you are using a vector that has GST or MBP, using the T7 forward primer will also amply the tag and your insert.
Good luck,
Joe
My experience is the same as Joe's. Yes, T7 forward and T7 terminator oligos work well as a primer pair for amplifying cDNA inserts.
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