DNA fragment Isolation from Low-Melting Temperature Agarose Protocol

[domba]

DNA fragment Isolation from Low-Melting Temperature Agarose Protocol

Based on Favre, D et al. 1992. Biotechniques vol. 13

1. Cut out slice containing DNA, smallest size possible.

2. Estimate volume and double with TE (10 mM Tris-HCl, pH 8.0/1 mM EDTA). Melt at 65 C 5-10 min.

3. Add 1 volume Tris-buffered phenol at room temperature, mix by inversion.

4. Spin 3 min at 10-12k rpm and transfer aqueous phase to new tube. Phenol extract again.

5. Spin as in (4) and transfer aqueous phase to new tube containing 0.1 volume 4 M LiCl. Mix by inversion; a white precipitate forms immediately. Place tube on ice 2 min. Spin as above, 3 min.

6. Transfer to new tube, leaving transparent pellet behind. Add 1 ul carrier (glycogen) and precipitate with 2.5 volumes cold ethanol. Mix, leave at -70 C 5-10 min, and spin as above, 10 min. Wash pellet with 1 ml 70% ethanol, dry under vacuum, and resuspend in 10-20 ul water or TE.

[rajyalakshmi]

Dear all,
I am very happy to get in touch with the forum.Since I teach molecular biology to undergrad and postgrad students I need many simple and clear protocols to follow in my labs.The protocol that I saw today is very nice,let me try it out.
thanks and regards,
Rajyalakshmi,Ph.D.

[kiki06]

Wow. Great stuff. I am happy to see everyones posts are very useful.

Welcome to the forum Rajyalakshmi!

Please tell us how it went.