Gel elution of a band - (Jan/18/2006 )
Hi all, I have a problem with a band I need to elute from a gel to seperate it from other DNA.
I'm told not to use colum/gel extraction kit and it seems there is no gel electroelution chamber available here too. The band is several kB big so no zentrifugation elution possible too.
Are there any other methods availabe?
Thanks.
Edit: I got someone right now telling me what is possible too.
Take the gel on the carrier under UV, cut a pocket around 4-6cm long BELOW the band out, put it back into the chamber but with buffer only reaching the gel surface, fill the pocket by pipette with buffer. Than let it run ca. 30 seconds at 200V. Pipett the buffer into an eppendorf tube and put it under UV to see if it is glowing.
To elute a piece of acrylamide gel, i use a 0,5mL eppendorf with a small hole. I fill this hole with glass wool. Then I put this tube into a 1,5 mL eppendorf. I freeze the piece of gel in liquid N2 (to disrupt the agarose) several times. Then i put the piece of gel into the small tube and i centrifuge . The agarose remains in the small tube and the DNA will be in the big tube. Then i do a phenol/chloroform and an ethanol precipitation.
This method works good ( better than kit)
This method works good ( better than kit)
Yes, my head of lab told me about this too when I mentioned centrifugation extraction but he says the DNA I want to elute is way too big for this to work. It would only break in fragments if anything.
Or what sizes do you elute that way maximum?
I'm told not to use colum/gel extraction kit and it seems there is no gel electroelution chamber available here too. The band is several kB big so no zentrifugation elution possible too.
Are there any other methods availabe?
Thanks.
Edit: I got someone right now telling me what is possible too.
Take the gel on the carrier under UV, cut a pocket around 4-6cm long BELOW the band out, put it back into the chamber but with buffer only reaching the gel surface, fill the pocket by pipette with buffer. Than let it run ca. 30 seconds at 200V. Pipett the buffer into an eppendorf tube and put it under UV to see if it is glowing.
How big is your band? What do u want to use it for?
I use a much simpler and user friendly technique to elute and reamplify PCR products. Under transillumination, i stab the band (better then slicing the entire band as the chance of accidentally including the neighbouring band is less) using a fine pasteur pipette so that a small cylinder of gel (with band) gets into the stem of the pipette. With a rubber bulb, i expel this small piece into a PCR tube, add 5-10 mic.lit of water and freeze-thaw it. Mashing the gel with a pipette tip will also do. Heat for a minute to 65C. Centrifuge at 10000 RPM and use a mic.lit of supernate for PCR.
Hi all, I have a problem with a band I need to elute from a gel to seperate it from other DNA.
I'm told not to use colum/gel extraction kit and it seems there is no gel electroelution chamber available here too. The band is several kB big so no zentrifugation elution possible too.
Are there any other methods availabe?
Thanks.
Edit: I got someone right now telling me what is possible too.
Take the gel on the carrier under UV, cut a pocket around 4-6cm long BELOW the band out, put it back into the chamber but with buffer only reaching the gel surface, fill the pocket by pipette with buffer. Than let it run ca. 30 seconds at 200V. Pipett the buffer into an eppendorf tube and put it under UV to see if it is glowing.
How big is your band? What do u want to use it for?
I use a much simpler and user friendly technique to elute and reamplify PCR products. Under transillumination, i stab the band (better then slicing the entire band as the chance of accidentally including the neighbouring band is less) using a fine pasteur pipette so that a small cylinder of gel (with band) gets into the stem of the pipette. With a rubber bulb, i expel this small piece into a PCR tube, add 5-10 mic.lit of water and freeze-thaw it. Mashing the gel with a pipette tip will also do. Heat for a minute to 65C. Centrifuge at 10000 RPM and use a mic.lit of supernate for PCR.
Any leftover of Agarose would be a problem in my case unfortunatelly.
I tried the gel elution method I had gotten from a friend, but there was no DNA in the samples left even with making sure I got no more band in my gel piece and it hadn't run into the opposite gel front too again. Maybe I have a problem with precipitation?
I have the fragment of course in TAE buffer and I used for precipitation 2.5V Ethanol and 0.1V Natriumacetate pH5.2. But is that working at all when TAE is a buffer already?
Anyone has experience with precipitating from TAE buffer?