4 replies to this topic

[kindmike]

After inserted a 300 bp foreign gene into topo 10 clone vector, use the following
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!!

[labrat]

Hi Mike,

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.

Quote

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???

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.

[kindmike]

Labrat, thank you!! I also think that there are two different plasmids may enter into the same cell. Do you think how to solve this problem?

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!!

[labrat]

I would suggest that you verify the insert by restriction cut or sequencing.  You know sometimes PCR can go wild. Because you are gonna do subcloning with the insert, it is very important at this stage to make sure.  Sequencing will also tell you if there are any base errors introduced by PCR.

[george@CASE]

quickest and surest is to have it sequenced but a double digest using an enzyme cutting once in the vector and once in the insert (preferably near the end) can aid you figure out the orientation.

Edited by george@CASE, 03 February 2005 - 04:34 PM.