Plasmid PCR - (Apr/11/2006 )
This is really a very basic question. Ive cloned a fragment of 700bp into PGemTeasy vector. After plamid preparation ive did a Restriction with EcorI and the insert popped out. To further confirm that it was what i was after ive did a PCR with one of the flanking primers plus a internal primer which should give 350bp. The plasmid preps that ive used (2) gave negative results. This is puzzling. Can the orientation of the insert inside the vector prevent the amplification? Or the PCR will always be positive if the primers recognise the insert?
when you say flanking, you mean vector specific right? if so, then yes, the orientation of your insert will influence the PCR (if it is the 'wrong' way round then you will essentially have 2 forward primers).....can you try the another set of primers on your plasmids?
Thanks for the reply.
No, when i say flanking i say the forward primer which was used to generate the initial band, combined with one internal primer which if the clone is ok should amplify a band of 350 bp.
if your primers are designed to amplify a shorter fragment, and as they anneal on the cloned fragment, tehre should be PCR.
But f you do pcr directly on minipreps, the plasmid is too concentrate. Did you quantify your plasmid?
how much template did you use?
Ive used a 1:1000 dilution and a 1:50 dilution and none gave positive pcr band...
i think best choice would be to quantify your miniprep. This quantification may serves as future standard for you minipreps conc. I mean once i quantify mineand it was roughly 250ng/µl. So applying the same protocol, i consider my minipreps are all 250ng/µl.
1:1000 and 1:50 is not very the same.
Considering my conc, and assuming a 1µl template, from 1:1000 it would be 0,25ng... not quite enough i think and for 1:50, it would be 50ng wich is too much and also inhibits pcr...