GENOMIC PCR - PCR PRODUCT FOR SEQUENCING (Mar/28/2009 )
I WANT TO GET INFORMATION THAT MANY KITS ARE AVAILABALE IN MARKET TO ISOLATE DNA FRAGMENTS FROM PCR PRODUCT....BUT I NEED A MANUAL TECHNIQUE THATS HOW WE ISOLATE DNA FRAGMENT FROM PCR PRODUCT.....THANKS
Akram on Mar 27 2009, 11:53 PM said:
I am not quite clear what your question is. But if you are trying to isolate a single DNA fragment from a mixture of 2 or more fragments, a gel purification and extraction would solve the problem.
If you have just one product after PCR (one distinct band on the control gel), you can simply do a phenol-chloroform-extraction followed by ethanol-precipitation to get rid of all proteins and the buffer.
But this only works if the fragments aren't too small (>100bp).
I also use another method for gel extraction if I have very small PCR products (>70bp) because you will loose most of your fragment by using a kit with columns.
I cut out the gel fragment as usual and put it in parafilm (I usually make a bowl in a piece of parafilm with my thumb) and close the parafilm tightly without creating pressure on the gel fragment!!!
I freeze the gel fragment for a minimum of 1 hour at -20°C. Then I take a cannula to make a small hole in a corner of the parafilm packet and press the liqiud out with my fingers into a reaction tube.
Afterwards I make an ethanol precipitation with the liquid.
Sounds very strange, but it works.
mastermi on Mar 28 2009, 02:51 PM said:
But this only works if the fragments aren't too small (>100bp).
I also use another method for gel extraction if I have very small PCR products (>70bp) because you will loose most of your fragment by using a kit with columns.
I cut out the gel fragment as usual and put it in parafilm (I usually make a bowl in a piece of parafilm with my thumb) and close the parafilm tightly without creating pressure on the gel fragment!!!
I freeze the gel fragment for a minimum of 1 hour at -20°C. Then I take a cannula to make a small hole in a corner of the parafilm packet and press the liqiud out with my fingers into a reaction tube.
Afterwards I make an ethanol precipitation with the liquid.
Sounds very strange, but it works.
An alternative to this parafilm-freezing techinque, which sound quite fiddly, is to cut the band from the gel and freeze it with liquid N2 if you have access to some. Easier and faster as it freezes imediately. You can then mash your frozen agarose-DNA in a mortar, and proceed to phenol-chloroform extraction.
almost a doctor on Mar 30 2009, 02:19 PM said:
mastermi on Mar 28 2009, 02:51 PM said:
But this only works if the fragments aren't too small (>100bp).
I also use another method for gel extraction if I have very small PCR products (>70bp) because you will loose most of your fragment by using a kit with columns.
I cut out the gel fragment as usual and put it in parafilm (I usually make a bowl in a piece of parafilm with my thumb) and close the parafilm tightly without creating pressure on the gel fragment!!!
I freeze the gel fragment for a minimum of 1 hour at -20°C. Then I take a cannula to make a small hole in a corner of the parafilm packet and press the liqiud out with my fingers into a reaction tube.
Afterwards I make an ethanol precipitation with the liquid.
Sounds very strange, but it works.
An alternative to this parafilm-freezing techinque, which sound quite fiddly, is to cut the band from the gel and freeze it with liquid N2 if you have access to some. Easier and faster as it freezes imediately. You can then mash your frozen agarose-DNA in a mortar, and proceed to phenol-chloroform extraction.
I thought that you wouldn't be able to get completely rid of agarose during phenol-chloroform extraction. But you never had problems with ligations after this procedure?