blunt end cloning
jim at U.ARIZONA.EDU
Fri Aug 30 08:45:33 EST 1996
Here is some protocol i used to use for blunt end cloning..
1) prepare insert with blunt end
( you can get it with pfu polymerase
or treat 1ul of klenow enzyme in pcr mixture according
the procedure accompanied by that enzyme..
2) cut the vector ( any vector will be fine once it has blunt end
restriction site , but i use the pGem 7zf with sma I )
gene clean it
3) prepare ligation mixture
( i usually use the same ammount of vector and insert
, not in the volume, but in the mass. i always quantity them
with ethidium bromide in agarose gel )
ligation buffer 2ul
water to 20 ul
4) incubate overnight at 22 C
5) before transformation, heat the mixture 65 C 20 min
6) add 1 ~ 2 ul of smaI and incubate 1 hour at 30 C
7) use them for transformation..
you can get much more white colony then blue one
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