How should I check my colonies? - PCR check is not working...:( (Jan/29/2006 )
ok im beginning to think that ill never pass to the protein expression stage...im just stuck with ligation... ...anyway....i have purified the gel slices as I have explained in another thread my PCR gel slice was weird (too big) and my extraction was not ideal...but when digesting this purified PCR with some enzyme that cuts in the middle of the insert (control digestion) i got my bands...so I suppose that my extraction was ok...
So I ligate and electroporate and get many beautiful colonies on my plate and one bad colony on the negative control plate (no vector plate not no insert) ....contamination i suppose...anyway i thought that all those colonies cant be just contamination....so I check 6 colonies with PCR...all are negative and my positive control is negative too.... so possible reasons i guess;
1-new polymerase which we bought now (fusion) so the reaction conditions that I have used before are not suitable anymore....
2-all colonies have no insert and my positive control is dead (plate is third month in the fridge)
so what do I have to do? do lysis for all the colonies and digest to see the insert? ...or just forget about it and do another ligation... but colonies on the plate are soooooooo beautiful...i just have the feeling that they have the insert but i cant detect it....sorry for the long post...
first you should have your positive control working again.
a nice way to have a build in positive control, if you chose the primers in a way that without template you end up with something like, let's say, 200 bp and with you insert than it's much more. if you can not detect any band at all, you know the pcr went wrong
Well if your positive control is not working, its not an issue of the ligation but an issue of the PCR. I agree with Kersten, get your positive control working. If the plate in the fridge is too old, streak out some new stuff. Also, how much colony are you adding to the PCR, too much can inhibit the PCR. You really need to just lightly touch the the colony with a tooth pick and add it to the PCR. It would be silly to re-do the ligation if you can't screen for whether its worked or not. Sort out your screening methodology before re-doing anything.
I can't believe you used a three month old plate as your positive control for colony pcr.
I would not use a plate that is older than two to three days for colony pcr. I bet all those colonies on the pos control are dead...
Prep the colonies you had (all six of them) and do a restriction digest to check for you insert.
hello all again.... thank you so much for your replies....i have changed PCR program a little bit and used freshly steaked colonies and got many positive clones!!!!!!! I cloning...
N.B. fusion polymerase concentration that ive used was 1unit/ul.....i think its too much but that is what molecular cloning says....how much do you use?
Use the minimum (0.5 U to 1.0 U). And don't throw away your money on a proof-reading polymerase when doing this. Buy the cheapest taq you can find with a little bit of detergent in the MM (tween 20, DTT, Triton-X100 etc. all work) and you should be set.
I find that it is much easier to amplify high copy number plasmids than low copy number plasmids.