DNA Fragment Purification from Polyacrylamide Gels Protocol

DNA Fragment Purification from Polyacrylamide Gels Protocol

Step by Step Isolation of DNA From Poly-Acrylamide Gels

1. To start, pour a vertical poly-acrylamide gel using TEA buffer.  Note that a 4 % non-denaturing gel is best for most DNA applications.
2. Run the DNA fragments. 
3. For most DNA fragments greater than 500 bp you should run the xylene cyanol dye to the bottom of the gel.
4. Stain the polyacrylamide gel for one hour in buffer with 1 ug/ml ethidium bromide
5. Cut out your DNA bands of interest using a scalpel and a UV box.
6. Transfer the DNA bands to 15 or 30 ml Corex tubes.
7. Add TE buffer to tubes - for a 15 ml tube add 3 ml of TE buffer. For a 30 ml tube add 7 ml .
8. Wash your tissue homogenizer probe first with DNAse-free water, then EtOH both before and after use.
9. Grind the polyacrylamide gel band piece in a tissue homogenizer for 10 minutes. Keep things cold with ice.
10. Place the tubes at 4 degrees overnite (O/N).
11. Spin down the polyacrylamide particles in a centrifuge at 10000 rpms for  10 minutes at room temperature and swing out rotor.
12. Carefully pipet off supernatant into a fresh Corex tubes.
13. Ethanol precipitate the DNA to isolate.