Hello,
After cloning, we chose the white colony from IPTG and Xgal agar, then cultured in LB broth and do PCR from that broth. We found the bands for our inserted gene, but not for plasmid. We use
pMD18-T plasmid and primers for plasmid is M13 Fwd and RV-M rev as in the protocol of manufacturer.
our pcr condition is
95 C - 3 min
95 C - 30 sec
52 C - 30 sec
72 C - 30 sec for 35 cycles
72 C - 5 min for final extension.
Any suggestions and advice are being waited for.
Thank you.
Lynn
Sometimes colony PCR gave us false positive result. If you found so, just change primers. On the other hand, not use too much bacterial as template.
Qingbao Ding
Thanks for your answer and advice, but, when we do colony PCR, we use neagtive control, so, it cannot be false positive, I guess. And also, we use only 1 ul from E. coli broth. The turbidity is also not too high. I need more suggestion, please.
Lynn
It isn't clear what you mean that you didn't find the band for the plasmid. What band were you expecting to find? Shouldn't those primers just give you the insert band?
Michael J. Benedik
Hello, Professor,
We would like to know if our desired gene is inserted into plasmid or not to do cloning. So, we use two primer pairs to check the plasmid and to check our gene.
If our gene is inserted into plasmid and if we use the primers for plasmid, the band will be the size of some sequences from plasmid+ our gene.
with best regards
lynn
Hi. Sis Tin Mar Lynn. In your question, you mean you want to know whether your gene is inserted into the plasmid or not, right? I am not sure about the procedure you did for this experiment (may be colony PCR for checking the correct insertion). I will briefly write down the procedure that I have done for successful insertion of the gene into the plasmid.
(1) Desired gene: A (PCR product)-size
(2) Plasmid
(3) Restriction enzyme digestion of the Plasmid (the enzymes choose for cutting the plasmid should be compatible with the desired gene)
(3) DNA Ligation (to get Plasmid+A)
(4) Transformation (Competent cell solution + ligation reaction solution)
(5) size screening for correct ligation and insertion (not colony PCR, but just mixing the colony with the solution from the size-screening kit) -> gel electrophoresis --> select band (Plasmid+A) (higher band than plasmid size)
(6) purification of the selected band --> check insertion : restriction enzyme digestion to confirm the correct insertion (Cut check) --> gel electrophoresis (1: control= plasmid only, control=plasmid+A (uncut sample), and two correct bands for plasmid size and gene size)
(7) Finally, subculture the correct insertion clone --> extract Plasmid DNA (Plasmid with desired gene) .
Hope this procedure and information will helpful for you.
Dear Lynn
i think that your resulta are right.
The PCR band depends from the primer that you are using and looking to the map of your vector
https://www.genomics-online.com/vector-backbone/37/pmd18-t-simple/
using M13 Fwd and RV-M you amplify the region of the insert.
If you would like to amplify also the vector you have to desing 2 different primers annealing in the same region but in the opposite direction but i don not think it is useful. Your result it is already prove that an insert of expected size is inserted in your vector.
YOu can find more guidelines about PCR colony screening and primers on my blog: PRoteoCool ( http://proteocool.blogspot.com/)
ProteoCool n°4 (PCR Colony screening) at PAGE 1
ciao
MAnuele
I think we are all a bit confused about your results. Using the primers you list there should be a single band amplified which is the insert plus a small amount of flanking region from the vector so the band would be slightly larger than the insert. For plasmids without an insert, that primer pair will give you a very small amplification product that you probably can't see on your gel. As Manuele Martinelli points out, you would need a different primer pair if you want to amplify part of the vector only, but there is really no reason for doing so.
Michael J. Benedik
Thank you so much for your answer. I am sorry for confusing question. Now, we have already solved that problem by changing competent E. coli cells. Competent cells had some problems.
with best regards
lynn
- Badges
- Science method