linearized vector purification from TAE agarose gel - (Feb/29/2008 )
I am using 1.0 ug of a 9.1kb vector that has been linearized (restriction digest) in a 0.8% TAE low melt agarose (Seaplaque) gel purification. Presently, I am using the GFX PCR DNA Gel Band Purification kit (#27-9034-70) which melts the agarose slice (65 degree) in a capture buffer ---- runs it through a spin column ----washes with wash buffer---- elute with autoclaved millipore water (30ul). The problem is that I am loosing 85-95% of my sample----is there a better kit or way of purifyng this amount of DNA?
have you tried to warm up the water before elution?
Maybe you could try 1xTE or 0.1x TE for elution instead of water (when I checked the pH of our MilliQ water, it was a way below pH7).
One other thing is to start with more DNA material (2-3 ug)??
We use here Qiagen Gelextraction Kit (500). You can extract DNA up to 10 kb. Well, it is not the best one but okay.
inaddition to Zek's suggestion of using heated elution buffer, I would suggest that isopropanol be added to the gel melt /capture buffer. I would also recommend that more capture buffer be used... the gel has volume and this volume dilutes the capture buffer when it melts. If the capture buffer is diluted sufficiently the DNA will not bind to the silicon matrix of the column. RUnning the capture buffer/gel melt several times through the column...(3 times) should give the column sufficient chance to bind.
thanks for your input----I'll give it a try
Get the Qiagen Gel Extraction kit protocol and see what they do. One thing not mentioned so far is to add 10 uL of 3M sodium acetate pH 4.2 to your melted sample before you put it on the column.
thanks---I'm going to try it