can I detect colony for the insert with the cloning primers? - or clamp sequence will interfere? (Jul/23/2006 )
ok i did this before and it was ok, but this time i can't check the colonies (did only 1 time) by PCR even positive control is negative. My professor thinks that it is because my primers are not excatly the same sequence as the vector constructed (have clamp sequences) and it effects the reaction. ..what do you think?
if your + control is negative, then your pcr reaction is not working. if you used the same reaction conditions as you previously did, then it must be your primers that create problem. as far as i know, commercial vectors generally have primer sets such as T3, T7 or Sp6 surrounding MCS which allows you to detect any insert. or your vector must have a specific set of primers surrounding MCS. and these primers, supposedly, do not create problems with PCR (since they are also sequencing primers).
u may prepare fresh primer dilutions from primer stock (if you used primer dilutions prepared 2 months before or more), your primers might be degraded.
or if you do not have lots of colonies than you may grow your colonies, isolate their plasmids and do restriction analysis to detect any inserts.
for your prof.'s suggestion, i think, u may align your primer sequence against your vector sequence to see whether it has clamp seq. or not
If your primers were designed to confer restriction enzyme sites then I'll suggest you designer/use primers that are specific to the insert flanking regions on the vector.
thank you everyone! the clamp sequence is not similar to the vector sequence..... but only 3 bps could affect PCR? and what is better to order primers of the insert which are completly complementary to the insert sequence or order vector primers flanking the MCS? thanx again
i will prefere to order primer set flanking MSC. u may use your vector for cloning another insert sequence and use the same primer set to detect it.