Extraction of PCR product from gel - (Jul/09/2008 )
Need everyones help
How cud I extract a PCR product from a gel. I have tried using the basic step of cutting the specified area containing my PCR product. Then keeping this gel piece in a centrifuge tube 500 microlitre which is having a hole in it and also containing a whatmann filter paper No: 1 . Then placing this centifuge tube inside a eppendorf tube of 1.5 ml. Then placing this tube in a microcentrifuge and centrifuging it for 2 min.
The solution which fills the 1.5 ml tube is then added with ammonium acetate pH 5.2, and then precipitated with 100 % ethanol.
Sometimes this protocol works but sometimes it doesn't.
But either way it is highly tedious and also cause maximum EtBr contamination.
Can any one suggest another method for extraction I am now planning to use Qiagen kit for extraction of PCR product from gel. But there's a confusion , is it safe to use elution buffer for eluting DNA from the coloumn as in one of the article I have read that it is better to elute the DNA using distilled water.
Sallie
How cud I extract a PCR product from a gel. I have tried using the basic step of cutting the specified area containing my PCR product. Then keeping this gel piece in a centrifuge tube 500 microlitre which is having a hole in it and also containing a whatmann filter paper No: 1 . Then placing this centifuge tube inside a eppendorf tube of 1.5 ml. Then placing this tube in a microcentrifuge and centrifuging it for 2 min.
The solution which fills the 1.5 ml tube is then added with ammonium acetate pH 5.2, and then precipitated with 100 % ethanol.
Sometimes this protocol works but sometimes it doesn't.
But either way it is highly tedious and also cause maximum EtBr contamination.
Can any one suggest another method for extraction I am now planning to use Qiagen kit for extraction of PCR product from gel. But there's a confusion , is it safe to use elution buffer for eluting DNA from the coloumn as in one of the article I have read that it is better to elute the DNA using distilled water.
Sallie
You should probably use the elution buffer in the kit. Distilled water sometimes has a lower pH than is optimal and the DNA doesn't elute properly.
Hello,
what is your specific problems?...I somethink have a problem whit etanol precipitation for PCR product (specially for very small product). I have best results with 1 volume of Isopropanol 100% and 1/5 volume sodium acetate (3M) and 15 minutes at -80° C...then centrifuge etc.
Ciao
If the PCR reaction give you just one band I recommend don't extract from the gel (is tedious, low yield, et.) If the amplicon will be use for cloning or sequencing is better to use a column type purification (centricon, qiagen have good ones), etc. less hassle, don't loose the poly A so easily, quicker, etc.
The EB buffer from the kit is just very slightly buffered (10mM pH 8,5) water, and is best for the elution. I use buffed water pH 8 for all my DNA aplications, primer dilutions and so on, the high pH prevents degradation of DNA.