[bio.dros] Subcloning large PCR fragments for transformations!

Tamas Lukacsovich lukacs at fly.erato.jrdc.go.jp
Wed Mar 27 21:15:27 EST 1996


Dear Dr. Mudgapalli,

I am not familiar with the PCR KIT you've used but suppose it includes
unphosphorylated primers.
The blunt-end ligation of a large insert lacking 5'phosphate (resulted from
PCR reaction) is very inefficient. I suggest you to try one of the
following ways:
1.) to supply your fragment with 5'phosphate using polinucleotide kinase,
2.) using inside restriction sites of your fragment if it is possible or
3.) ligating linkers to your fragment before cloning (the large excess of
linkers may facilitate the blunt-end ligation).

Have a good luck!

Tamas.
-------------------------------------------------------
  TAMAS LUKACSOVICH, Ph. D.
  ERATO, Mitsubishi-Kasei Institute of Life Sciences,
  11 Minami-Ooya, 194 Machida-shi, Tokyo, Japan
  Tel:  (81) 427-21-2334
  Fax:  (81) 427-21-2850
  email : lukacs at fly.erato.jrdc.go.jp
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