having the wrong vector in my clone - (Nov/16/2009 )
Hi, everyone I have been trying to do my cloning for 3 months, but i couldn't get the clone. So here is my problem.
I'm cloning my insert into pET15b vector. I did a ligation and transform them into DH5alpha. There are lots of colonies grew on the plate, then i screen them with colony PCR using the T7 promoter and terminator primers, as well as with my insert primer. Apparently, the PCR with my insert primers showed me products, but none of the T7 primers showed me products. So i decided to extract the plasmid from the colony that gave me positive result in my colony PCR with insert primer. Then I did a PCR using the T7 and insert primers again with the plasmid I've extracted. There were no positive results for my insert primers, but they were positive result for my insert primers. What is happening here?? I should able to get positive results with T7 primers because im cloning into pET15b. If my insert is not cloned into pET15b, where has it cloned into???
PLEASE HELP ME!!!!!
Did you digest the extracted plasmid(s) to see if there's an insert?
Do the T7 primers give you a product on a "known-good" clone from another experiment (any insert cloned into pET15b)?
Did you do colony PCR right from the transformation plate, or did you pick them first to new plates and then do colony PCR after they'd grown?
Is there a copy of your gene (or a close homolog to it at the DNA level) in E. coli?
Thanks so much for replying. I have digested the extracted plasmid and there is an insert in my plasmid. The T7 primer always gives me good results, i used them as my positive control. i did my colony PCR right from the transformation plate. I have no idea what is happening.. The T7 primers should be fine, but it doesnt give me positive result when i do the PCR.
Then i decided to do another PCR using different combination of the primers.
1. T7 promoter & Insert Reverse
2. T7 terminator & Insert Forward
3. T7 promoter & T7 terminator
4. Insert Forward & Insert Reverse
The PCR results showed the first (1.) and forth(4.) primer combination works, but the second(2.) and third(3.) does not work. It seems like those combinations with T7 terminator primers is not working. But my positive control for T7 promoter & T7 terminator works perfectly well. what is the reason???
I have sequenced my clone using T7 promoter and terminator primers. With the T7 promoter primer the sequencing works perfectly well and there were no mutations with my insert. However, the T7 terminator primer did not show any sequencing results, no results were obtained, no signals. Then i looked at the sequence obtained from the T7 promoter primer, there is something weird about the results. The insert sequence was good, but the sequence after my insert is not my pET15b vector sequence. Shouldnt the sequence be pET15b after my insert? I digest my insert and pET15b with NdeI and XhoI, shouldnt the sequence be pET15b sequence after the XhoI?? And my insert is not a PCR product, i clone my insert into pGEM then digest them.
What is happening???