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clonning gel purification


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#1 Isb

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Posted 22 September 2004 - 03:07 PM

I am tyring to clone a fragment of L chain ~ 600bp from a plasmid (4.2 kb) into a vector that is 7.2 kb. The vector has a different L chain (also 600 bp) which is flanked by XbaI and Sac I. I digest both the vector and the plasmid with XbaI and Sac I. So when I double-digest(both enz. bought from Roche and have 100% activity in Buffer A) the vector , I get two bands, at 6.6kb and 600bp. When the plasmid is digested in the same manner, I get a band at 3.6kb and another at 600bp.
My problem is when I do a gel purification using Qiagen's Gel extraction kit(for the vector) and then proceed with ligation(using T4 from NEB, RT for 10 mins and then inactivation at) 65C for 10 mins) and then transformation, I do not get any colonies.
However, if I donot gel purify but do a clean-up after my digestion and then proceed to ligation I get colonies. The problem here is that I might have some colonies that have the religated original vector while some may be the vector containing the L chain of my interest in the vector. I have no way of knowing. I know this much that when I add ligase to a tube that has the digested vector only (meaning both the 6.6 kb and the L chain originally from the vector since I didnot gel purify), I get a few colonies. When to another ligation tube that has the same contents, I also add the L chain of my interest(PC amplified), I get many many colonies.
So I think, gel purification is my problem.... can somebody pls. give me some ideas .....suggest some alternate gel purification kits..etc.......any help will be highly appreciated.

Thanks a lot

#2 turtle

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Posted 22 September 2004 - 03:35 PM

are you losing the fragment when you gel purify? Or not getting it clean enough? There are LOTS of gel purification kits on the market.

If the problem is that you are losing your band, you might consider spinning the gel slice in the filter basket from the kit without dissolving it in binding buffer, then doing an ethanol precipitation of the effluent (the liquid in the tube after you spin). This method results in basically 100% recovery of your fragment, but it won't be as clean as if you actually bound the DNA to the membrane and re-eluted in Tris.

Also, if you are using water to elute your fragment from the column, rather than the elution buffer supplied with the kit, make very sure the water has a pH of 7.5 or above! Otherwise it won't work.
Turtle




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