Dear All,
Please help me out, I am trying to transform three plasmid pGE5X3, pWB980 (an E.coli-Bacillus shuttle vector) and a control plasmid pUC19 using the NEB 5-alpha Competent E.coli (High Efficiency) comp cells. But I am getting colonies only for the pGEX vector (I repeated 3 times) and other vectors are unable to transforms even including the control one. So anyone please suggest me what could be the region?
Thank you,
Ranjeet
Hi there,
Have you checked the actual quality of the content of your plasmid preps by agarose gel electrophoresis? I would suggest to prepare fresh plasmid and perform the transformation again. Be careful with the selection marker as they are not the same for the 3 plasmids.
1) You need to be careful with the selection marker. All 3 need to be different. It looks like pUC19 and pGEX3 have the same AmpR marker. pwB980 has KanR marker.
2) Origin of replication (ori) should also be different and compatible with each other. pUC and pGEX do not have compatible ori.
For compatible ori, refer
http://blog.addgene.org/plasmid-101-origin-of-replication
Dear Dominique Liger and Ramesh K Jha thanks a lot for your critical suggestions.
Regards,
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