fragment isolation w/o glass milk?

John Brunstein brunstei at UNIXG.UBC.CA
Mon Aug 8 13:28:42 EST 1994


	Actually, someone (sorry, but I don't remember who) posted the 
'easier version' that you are looking for on this newsgroup just a few 
months ago.  I took his protocal  and modified it very slightly myself 
and have had good luck.  It goes as follows:
	(1) Silanize a batch of glass wool (the 'soft' grade is easier to 
work with than the 'stiff' grade, but both work OK) by putting in a 
beaker, adding a few ml of silane (anybody out there tried Rain-X for 
this?) and slop it about to get the glass wool all wet.  I then put the 
beaker in a 60C oven for a few hours to dry the wool (original post 
suggested vacume dessicator overnight, but I'm too impatient).

	(2) Cut the tops off a batch of small (0.5 or 0.6ml 
microcentrifuge tubes) and use a 22 gauge needle to poke holes through 
the bottoms.  

	(3) Take a small pinch of the silanized glass wool and squash it 
into the small tube.  I use the plunger from a 1cc syringe with the 
bottom rubber part broken off to stomp the wool down.  You want ~4-5mm 
height of the wool.

	(4) Cut the cap off a large (1.5 ml) microcentrifuge tube, put 
the prepared small tube into the big one (it should catch on the rim, 
such that there is ~100 ul free volume at the bottom of the assembly).

	(5) Now the easy part---just cut your band out of the gel (as 
small a slice as possible), put the slice into the premade assembly 
(don't worry about crushing, etc; just pop it in) and spin 12000 x g for 
2 minutes.
	
	(6) Your fragment will be in the aqueous phase in the bottom of 
the big tube in 20-100 ul of solution.  For a band of any reasonable 
strength, I usually find the best results for cloning is just to use a 
few ul of this directly in the ligation.  For some reason, this works 
better in my hands than doing a ppt. of the extract and resuspending in a 
smaller volume, but then again maybe I'm just a klutz.

	I have used this successfully for a few months now (~40 
isolations?) on bands from 80bp to 9kb, with gels of between 0.9 and 2.0% 
normal agarose.
	Hope this answers your questions, and my thanks to the original 
poster whose protocal this is based on.












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