I did gel electrophoresis after PCR and get the correct gene size (2.3kb). Then, I purified it from gel and ligated it into pJET1.2 with blunt end ligation, transformation, miniprep and sequencing. But, after sequencing, I only get the first 300 or 700bp of the gene. I also did colony PCR with gene specific forward primer and vector specific reverse primer and could not get the right size. I got around 700 bp the same as with the sequencing result. Anyone who have suggestion to solve this problem?
Hello Yeshambel,
One possible cause could be if the sequence of your insert contains one or more long direct repeats that could foster excision by recombination. Choosing a recA recipient strain may help in that case. Also, I would advise you to test 10-20 further colonies by performing colony PCR, which is easily done (take care of patching each of the tested colonies on a separate plate with a toothpick before dipping the toothpick into 10 µl PCR mix with all goodies except template DNA). Once you find a couple of colonies that yield a PCR fragment with the right size (2.3 kb), make minipreps and send one for sequencing (I keep the second one in case the first one happens to carry a spurious mutation, which occurs rarely if you use a high fidelity DNA polymerase to prepare the fragment to be cloned).
Hi, Yeshambel, did you solve your problem? I am also having the same problems with promoter amplification.
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