I have tried preparing genomic library construction from MDS69 strain of E.Coli, transformed with Keio genomic DNA (blunt end) using the PSG1143 vector. But unluckily the transformation efficiency was extremely low, even negligible. Can anyone tell me the possible reasons and suggestions for improvement will be highly appreciated.
Transformation efficiency depends on multiple factors. The major factors are,
1. Competence of E. coli cell.
2. Size of the ligation product / vector.
What method you are following to prepare competent cell?
Have you checked the transformation efficiency with the vector alone?
Thanks Bhisma Narayan, I have used Electroporation and the competency of the cell is not excellent but good. The size of the insert is b/w 10-15 kb. and the vector size is 6 kb.
Any idea what to focus on ?
Also depends on the E coli strain that you are using. Go through this paper (http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047654), they have transformed large DNA into bacteria. All d best.
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