Colony PCr - (Oct/10/2007 )
Hello, I know that this topic has been extensively discussed in this forum... but for new people like me, who just about to do colony PCR, I'm still a little bit blur .. well, my question is: if you do colony PCR, what is the diagnostic band that you expect to see, is it your insert alone or is it with some other bands? secondly, let say, i decided to do colony PCR using one of the vector primer (T7 reverse) and one from my insert primer (forward primer), what will i expect to see? and how can we confirm that my insert is in the correct orientation?
thanks in advance for any explanation.
new beginner: spices
1) It really depends on your primers. You can use back your own primer or universal one. Usually Colony PCR will give you only 1 distinct band.
2) If you are using universal primer, just check the plasmid you had cloned together with the insert. Work that out and you will be able to find out the expected band size.
3) for orientation, you can only do it with sequencing. Cant confirm with colony PCR
I disagree -- we do PCR all the time to simultaneously detect the presence of an insert and it's proper orientation. So long as one primer binding site is vector borne, and the other, oppositely facing primer is insert borne, the only PCR that will work is if the insert is actually cloned into the vector, and it is in the desired orientation (aside from amplifying completely around the plasmid -- a possibility easily avoided by proper extension time in your PCR program, and also easily ignored if it occurs due to the size of the product produced relative to what you're expecting).
NOTE: forward primer meaning sequence is on (or same as) the sense strand. Also I'm assuming that the T7 is at the downstream of MCS. Then, as HomeBrew pointed out, you'll get :-
(1). ONE band for the construct that has an insert with correct orientation
(2). NO band for construct with an insert but wrong orientation (only happens with single RE "cut and paste"), OR construct without insert (self ligation)
(3). TWO bands (one higher the other at expected size) if there's incident of concatomer, but that'd normally be labelled as "anomaly" and ignored in my cloning. If, as HomeBrew pointed out, your PCR extension time is optimized, then you'll see only a band of expected size w/o higher band. (Safe to ignore this)
hello, thanks for all the reply.. but I am still 40% unclear (sorry, quite a slow learner... ), anyway, my using pET 101(5753bp) as my vector, insert comes in at 302bp, to make things easy, let say my insert size is 600 bp and the T7 reverse priming site start at 454-474 bp. so, what will i be expecting if the insert is in the correct orientation. Thank you
still confuse: spices