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After ligation,I found that my colony on amplicilin plate have not insert gene

ligation digestion

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#1 sagebook

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Posted 17 January 2013 - 09:31 AM

I digest gene(520bp) and pETduet-1 plasmid(5420bp) by BamHI-EcoRI, and then keep product in elution buffer(Nucleospin kits). Next step,I ligase both gene and pETduet-1 plasmid product by T4 DNA ligase at 22c for 3 hour and then transform into DH5a competent cells.The next day I found, there are 5-6 small colony on amplicilin plate(primary plate) and then check there colony by colony PCR, I found that my band(520bp) is weak(thin).Next step,I steak bacteria on plate(secondary) from colony in primary plate incubate 37c overnight.I check colony in secondary plate by colony PCR.I found,There are no my band(520bp).So I try extract plasmid from primary plate, I found that molecular weight band of plasmid are not about 5420-5900bp but are 3200bp.
My problems are....1. How did "insert gene" lost?(while there are colony on amplicilin primary plate and weak band in gel)
2. Why? molecular weight band of plasmid are not about 5420-5900bp but are 3200bp?
I don't understand T_T reply please.I'm worry.
Thank you very much

#2 bob1

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Posted 17 January 2013 - 11:45 AM

The weak band could easily have come from ligation mix that was plated out when you spread the transformed cells onto the plate.

If you only have a few colonies from a transformation, and your positive control transformation has lots, then there is probably something wrong with your ligation reaction. You should also try to transform appropriate ligation controls (e.g. cut vector +ligase, cut vector alone).




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