Team:Peking S/lab/protocol/DNApurify

__NOTOC__
{{Template:Http://2011.igem.org/Team:Peking_S/box4lab}}
{{Template:Http://2011.igem.org/Team:Peking S/background}}




<font size=6> <font color="#FFFFFF">Protocol</font></FONT>



{{Template:Http://2011.igem.org/Team:Peking S/protocal}}



=Protocol for DNA Purification from Reaction Mixture=
<html>
<a href="https://static.igem.org/mediawiki/2010/b/bf/Protocol_for_DNA_purification_from_reaction_mixture.pdf"target="blank"><img src="https://static.igem.org/mediawiki/2010/9/91/PKU_Adobe_Reader_Logo.jpg" width=20>download PDF version</a>
</html>
Here is a suggested protocol; this protocol can be used to purify a wide range of DNA fragments with recoveries of >80%. The bolded should be noticed for a nice DNA extraction.

1. Put EB (elution buffer) or ddwater at 65 degree water bathing.

2. Add a 3:1 volume of Binding Buffer to the reaction mixture (e.g., for every 100 ul of reaction mixture, add 300 ul of Binding Buffer). Mix thoroughly.

''' Check the color of the solution. '''A yellow color indica tes an optima l pH for DNA binding. If the color of the solution is orange or violet, add 10 ul of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.

3. Pour the solution to a fresh adsorption column. Centrifuge at 13000rpm for 1 min.

Pour off the liq uid in the collection tube.''' For critical samples,''' repeat the operation above.

4. Add 600 ul washing buffer (WB) before centrifugation at 13000 rpm for 1 min. Pour off the liq uid into beaker.

5. '''Centrifuge at 13000rpm for 10 min '''to spin the ethanol down.

6. Put the column into a fresh EP tube. If necessary air-dry the pellet for 10-15 min to avoid the presence resid ual ethanol in the purified DNA solution. Resid ua l of ethanol in the DNA sample ma y inhibit downstream enzymatic reactions.


7. Add 30-50 ul elution buffer (EB) to elute the DNA.

8. Get 5 ul of the eluted sample to identify with electrophoresis.



==Note:==
1. If a large amount of DNA is purified or if the volume of the binding reaction is greater tha n 1.5 ml increase the incubation time of the binding step to 15 min.



==References:==
*Current protocols in molecular biology