Cleaning DNA for in vitro Transcription

(version 1.0;  01/27/04)

 

Note:  This protocol uses toxic substances and should preferably be done in a fume hood.  If one is unavailable, work in a well ventilated area and use safety goggles until the DNA precipitation step.

 

1.    Bring volume of DNA solution up to 200 µl with TE or MilliQ H2O.
2.    Add 200 µl phenol[1] ­ vortex for 30 seconds.  Spin 3 minutes at 14k rpm.
3.    Carefully remove aqueous phase (top layer) leaving 5-10 µl behind.  Try not to remove any of the organic phase or white proteinatious layer.
4.    Add 200 µl of phenol/chloroform/isoamyl alcohol[2] ­ vortex for 30 seconds.  Spin 3 minutes at 14k rpm.   Carefully remove aqueous phase leaving 5-10 µl behind.
5.    Add 200 µl of chloroform[3] ­ vortex for 30 seconds.  Spin 3 minutes at 14k rpm.
6.    Carefully remove aqueous phase leaving 10 µl behind.
7.    Precipitate DNA by adding 20 µl 3M NaOAC ph5.2, and 500 µl EtOH
8.    Vortex and sit on benchtop for 5 minutes.
9.    Spin at 14k rpm for 10 minutes.
10. Remove supernatant and wash with 500 µl 70% EtOH.  Let sit at RT for a few minutes before dumping wash.
11. Air dry for 5-10 minutes.  Resuspend in 50 µl sterile MQ H2O.  Run 1 µl on gel to check yield.

 

Alternative method: remove RNAses by using QIAquick PCR purification (#28104).  Add 5 volumes PB buffer.  Load onto column.  Wash 5 times with PE wash.  Spin twice after last wash to remove all of PE wash buffer.  Elute DNA in 50 µl EB buffer.

 

 

 

 

 


[1] Tris-saturated Phenol (Roche #100995)

[2] Phenol/Chloroform/Isoamyl alcohol (Roche #101001)

[3] Chloroform (Fisher #C606-1)