For past one month, I have been struggling with a ligation reaction. I have been trying to insert a ~7kb DNA fragment into ~11kb plasmid and both them have been preparation by restriction enzyme (NruⅠ-HF and ASCⅠ from NEB ). Then the vector and insert have been ligated by T4 DNA ligase with PEG 4000 under 22℃ for one hour. The vector has been dephosphorylated well. The molar ratio of the vector and insert is 1:3. However, I got a unexpected result that the recombinants are only 4kb. I could not understand the result hitherto. Thus far I have not been able to obtain a clone consisting of the 11 kb fragment and the 7kb fragment. Does anyone have any suggestions?
Are you using chemically competent cells or transforming by electroporation? Large DNAs are notoriously inefficient at transforming chemically competent cells. You might also consider making a new prep of your vector. You might have a stock of vector that is very slightly contaminated with a deletion variant that is 4kb, and the high background of transformation by this deletion plasmid is masking your real clones. You could test for this by doing an unligated vector-only transformation.
It's the template of PCR and the template is a ~4kb plasmid.I purified it by PCR Clean-up kit.
Thank Imran Khan and Michael J. Benedik.
PCR cleanup removes small molecules but not necessarily template DNA. You could try a brief DpnI digestion of the PCR product before ligation or gel purify the PCR product.
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