About identify the inserted gene?
Posted 27 January 2005 - 06:00 PM
two ways to identify the inserted gene;
1. Used EcoR1 digesting and got a right size of the inserted gene.
2. Used M13 forward primer plus forward primer of the inserted gene, and M13 forward primer plus reverse primer of inserted gene doing PCR, Why all got the bands of amplification???
Or try M13 reverse primer instead of the above M13 forward primer, also got the both of bands amplified, who can tell the reasons?? Thanks very much!!
Posted 27 January 2005 - 10:12 PM
The best way of identifying insert is restriction digestion because PCR sometimes gives false positive. For PCR, you can use M13 forward + reverse. Since you don't know the orientation of the insert.
You mean you used the primer pair to amplify the same template? If yes, one of band may be contamination.
There is the possibility that the colony you picked is not derived from a single clone, or two different plasmids may enter into the same cell.
Edited by labrat, 01 February 2005 - 10:02 PM.
Posted 01 February 2005 - 07:37 AM
I have analyzed 38 clones through isolating their plasmid using miniprep kits, and do the above PCR. the result is same with the first one above I posted.
Do I need to redo the transformation? If I can comfirm the insert gene and right orientation in Topo cloning vector, I will use double digest to ligate another vector, then do transformation. now I am sticking in Topo cloning. Do you or anybody else have some suggestions? Thanks a lot!!
Posted 01 February 2005 - 10:01 PM
Posted 03 February 2005 - 04:33 PM
Edited by george@CASE, 03 February 2005 - 04:34 PM.