Posted 14 May 2011 - 10:53 AM
Then I went back to the entry plamsids and found I couldn't PCR them out either. But they were amplified from the original e coli containing the plasmids confirmed by sequencing. The odd thing is most of those plasmids gave right bands when do RE cutting. I think they might be good plasmids but somehow couldn't be PCRed or do recombination. I am wondering if anybody got any ideas why they behaved like that? Is it possible that the potential tight packing prevents the PCR and recombination? Thanks a lot in advance for your input.
Posted 16 May 2011 - 04:51 PM
Posted 18 May 2011 - 07:48 AM
The entry clones have all been sequenced before and they were good at that time. I believe they are probably still good because RE cutting worked for most of them. I just don't know why PCR didn't work. PCR conditions should be all right since some of the clones can be PCRed out. The primers should also be OK because they are either M13 or tested gene specific primers. Most problematic clones have YFP or GFP in.
Is there any possibility that plasmid could self cut the insert off?
I used those clones for three-way gateway reaction. What I obtained before were kind of false positive. By false positive, I mean I got colonies, but they may only have two inserts in. Its wired but seems so by sequencing. Anybody has any idea what happened and how to improve the situation? Thank you very much!
I have few questions in my mind regarding your problem. After preparing entry clones, did u check them by PCR prior to sequencing? Are you using M13 primers or the gene specific attB primers to PCR check your entry clones?