blunt end ligation problems

HY Yang yang at tifton.cpes.peachnet.edu
Sat Aug 17 21:39:07 EST 1996


Hi- I don't konw what insert you are cloning. Use TA-cloning vector for 
PCR product, or polish the PCR product by T4 DNA polymerase using dNTPs.

Good Luck


H.Y Yang

ahouse at hydra.rose.brandeis.edu (Jeremy C. Ahouse) wrote:
>Hello group,
>   we are having a hard time with blunt end ligation. We are polishing
>ends with pfu polymerase. Any suggestions given that? How sensitive are
>ligases to salt concentration? I have read about the use of PEG and seen
>reference to a protocol that changes the concentration during the ligation
>reaction (first high conc favoring insert+vector, then low favoring
>circularization). Any thoughts?
>
>   Thanks,
>
>   Jeremy C. Ahouse
>
>-- 
>Jeremy C. Ahouse, Biology - Brandeis University
>Waltham, MA 01432-1515
>617.736-4954 (lab) 617.736-2405 (fax)
>ahouse at hydra.rose.brandeis.edu (email)





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