the problem on plasmid ligation and transformation

ygzhou ygzhou at 263.NET
Wed Aug 11 03:55:14 EST 1999


Hello Dr.,

ÐÇÆÚÒ», 1999Äê8ÔÂ9ÈÕ, you wrote:

DDZ> Hi,

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

DDZ> Zha

i also met this problem before. the reason may be in the step of enzyme
cleaving the pcr product. Generally speaking there are at least
three bases to protect the enzyme cleaving postition of PCR product.
i could not  clone one 2kb pcr product in the plasmid for many times.
after several failures i seek another method by blunt terminator
ligation. in the end by sequencing the target product, i know that there are only
two bases for protection. if possible , i recommend that you firstly
clone the PCR product in one small plasmid such as puc19 by blunt
ligation. then cleave the target product by RE and clone to the biger
plamid of 6 kb. wish u good luck





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