3 replies to this topic

[alex]

I got trouble in extracting genome CNA from a gram negative bacteria, the extract was very sticky. My procedure: lysis with proteinase k, extract with phenol/cheoroform 3 times, EtOH precipitate, dessolve in water.  Any suggestion is appreciated. thanks!

[Frederic]

Hi, here is a protocol for extraction of gDNA from E. coli

-Take 1.5 ml full grown culture and centrifuge 2 min
-discard supernatant
-resuspend pellet in 567 µl TE by repeated pipetting
-add 30 µl SDS 10% and 3 µl Proteinase K (20 mg/ml)
-Mix and incubate 1 hour at 37°C (waterbath)
-add 100 µl 5M NaCl and mix
-add 80 µl CTAB/NaCl and mix
-incubate 10 min at 65°C
-add an equal volume of chloroform/isolamylalcohol
-spin 5 min at 14.000 rpm at 4°C
-take carefully supernatans to new tube (no protein interface)
-add an equal volume of phenol-chloroform-isoamylalcohol
-spin 5 min 14.000 rmp 4°C
-transfer supernatans to a new tube
-add 0.6 volume isopropanol and shake tubes back and forth until you see the white DNA strands appearing
-spin 1 min 4000 rpm room Temperature
-carefully take of supernatant
-wash pellet by adding same volume EtOH 70%
-respin same conditions
-take off supernatant
-air dry pellet
-redissolve pellet in 100 µl TE overnight at 4°C
-measure concentration and freeze

This always give a high yield and a good purity (more than 1000 ng/µl). If you have problems, mail me
frederic.vandemaele@agr.kuleuven.ac.be

succes

[SamBrilliant]

Extract being sticky is always a good sign, at least you have lysis. Dilute the 'sticky extract' with 4 volumes 10 mM Tris.HCl buffer pH 7.7 and go ahead with procedure as before.

Good Luck

Sam Brilliant
dstf@doctor.com
Ph.D. Rescue Team
A division of DSTF-Global
http://www.dstf.bigstep.com

[rhodadg]

I've used the protocol described by Frederic but for a large scale extraction and I encountered the same problem with viscous extract. The yield was very high but  the genomic DNA was difficult to cut.  I cleaned it up using the column from the DNAeasy kit from Qiagen.  Just treat your extract as the starting material and follow the protocol as outlined.  I still had fairly high yield and the DNA was no longer viscous and was very clean.