blunt end cloning

[Jin-seon Im]

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 )

	eg

	insert 7ul
	vector 5ul
	ligation buffer 2ul
	ligase 1ul
	smaI   1ul
	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


cheers

jsim