the problem on plasmid ligation and transformation

Robert De Lisio r.delisio at worldnet.att.net
Sun Aug 22 23:31:12 EST 1999


The original posters problem sounds like he is simply having difficulty in
getting efficient cutting with his enzymes.
In light of this I have two questions:
1. What enzymes are you using?
2. How many additional bases did you add to the 5' end of your PCR primers?
Most enzymes require 4 or more extra added bases. It may be that the number
of bases you are using does not allow for efficient cutting for one or both
of your enzymes. I routinely add 6 bases just to be on the safe side but you
may add more if needed. Consult NEB's website for the precise number for
your particular enzymes. As an example, the restriction site is in caps in
the oligo below. The added bases are 5' of that. If you do not have enough
added 5' bases your enzymes will not cut the amplicon even thought it
contains the correct restriction site.

5' gatcgtGAATTCgatgcctagctacgacgt 3'

Hope this helps.
Bob

"Dr. D. Zha" wrote:
>
>  Hi,
>
>  I have met an old problem, when I try to clone the PCR product which is
>  cut by two different enzyme into a plasmid, it does not work. The length
>  of the plasmid is around 6 Kb and the insert is around 1 kb, and recover
>  the DNA bands after digestion from agarose gel by kit, and using T4 DNA
>  ligase which bought from Boheringer, and the competent cell was prepared
>  by the method of RbCL which I used the my former laboratory quite
>  successful, and the transformation of the cell can reach 10 to 6 cfu/ug
>  the plasmid, would please help me to solve the problem, I appreciate any
>  helpful suggestion, Thanks!
>
>  Zha







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