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gel extraction protocol for small amount of starting material
3 replies to this topic
Posted 17 April 2009 - 10:59 AM
I am trying different protocols to extract small amount of DNA(100ng-500ng) from agarose gel. I have tried phenol chloroform extraction, didn't work for me. Anyone has a protocol that works? I am thinking to try mega-beads, but my gel piece is 300mg, too big for their column. Any suggestions?
Posted 17 April 2009 - 01:04 PM
I will try this:
1. Cut gel with the desired band on a low intensity UV with sterile sharp.
2. Put the cut gel in an epp tube and freeze at –70o C for 15 min.(Max 24Hrs)
3. Melt gel on Heat Block (5 min,65C) & add eq vol TE sat Phenol. Gently crush gel with tip.
4. Vortex gently and freeze at -70°C for 15 min.
5. Thaw the tube and spin at RT for 5 min at 12000 rpm.
6. Take out the aqueous phase in another clean tube and add equal volume of Chloroform: Iso Amyl Alcohol, (24:1). Spin at RT, 12,000 rpm, 5 min.
7. Take out the aq phase carefully & add 3M Sodium Acetate (pH-5.2) so that the final conc of Sod Act is 0.3M.
8. Add twice the vol of prechilled abs ethanol. Mix well by inverting. Inc at -70°C for 30 min. (Max24 Hrs)
9. Spin @ 13,000rpm, for 10min at 4°C. Remove SN and wash the pellet with 70% pre chilled Ethanol(500μl). Mix well, inc at RT for 5-10 min.
10. Spin @ 13,000 rpm, 7 min at 4°C. Remove the SN, Air dry the pellet at RT and re suspend in 20/40μl of TE/MQ.