>Can anyone suggest a good DNA prep for 1-2 micrograms from chlamy?
>Either a ref. or a protocol would be appreciated. Does anyone else
>have trouble with polysaccharides in their preps?
>Thanks
>Hank Latorella
>SUNY Geneseo
>Geneseo, NY 144
PREPARATION OF DNA USING XANTINE
For transformant analysis and prep of DNA from wt cells
1. Centrifuge 5-15 ml of cells for 5 minutes at maximum speed using
table-top at 3000 rpm. Use
polypropylene 15 ml conical tubes.
2. Remove medium very well. Cells can be frozen at -70 C at this stage.
3. Add 2 ml of Xantine buffer and vortex the tube to resuspend cell
pellet.
4. Place the tube in a 65 C water bath and incubate for 40 minutes.
5. Centrifuge for 5 minutes as in #1. Collect supernatant to fresh 15 ml
polypropylene conical tube. Discard pellet.
6. Add 5 ml of 95% ethanol (2.5 volume ). Mix well. At this point tubes
can be stored in -70 C freezer.
7. Centrifuge 5 min. at 3000 RPM to collect DNA. Discard ethanol.
Centrifuge the tubes for 1 minute and remove remaining ethanol with
capillary tip. Remove all alcohol.
8. Resuspend pellet in 300 ml of TE buffer (or water). Transfer the DNA
solution to microfuge 1.5 ml tube.
9. Add 150 l of 7.5 M ammonium acetate. Mix well by inverting several
times.
10. Add 1000 l of 95% ethanol and mix well.
11. Centrifuge for 10 minutes in microcentrifuge. Discard supernatant.
12. Wash pellet twice with 700 l of cold 70% ethanol. Centrifuge for 30
sec after last ethanol wash to collect remaining ethanol from the
centrifuge tube wall. Remove ethanol with capillary tip.
13. Resuspend pellet in 300 l of TE buffer. Add 10 l of DNase free
RNase A and 1 l of RNase T1. Mix well and incubate for 30 minutes at
37 C.
14. Add 150 l of 7.5 M ammonium acetate. Note: Ammonium acetate must be
fresh, i.e., not stored more than 1-2 weeks. Mix well by inverting 3-4
times.
15. Add 1000 l of 95% ethanol. Mix well by inverting 4-5 times.
16. Centrifuge for 10 minutes at room temperature. Discard supernatant.
17. Wash pellet 2 times with 700 l cold 70% ethanol. Remove last drop of
ethanol as described above after last wash.
18. Resuspend pellet in 20-50 l of sterilized water. Store in -20 C.
Concentration of DNA is 2-5 g/ l.
Note: You should expect 750 g DNA from 10 ml of cells.
Note: Scale up DNA preparation by using multiple tubes not a larger
volumes.
Potassium ethyl xanthogenate (from Fluka cat # 60040)
Synonym: Carbonodithioic, o-ethyl ester. MW 160.3
RECIPE FOR XANTINE BUFFER:
Ingredient Amount/100 ml Concentration
Potassium ethyl xanthogenete 200.0 mg 12.5 mM
1 M Tris-HCl pH 7.5 10 ml 100 mM
0.5 M EDTA pH 8.0 2.0 ml 10 mM
NaCl 4.09 g 700 mM
Water to 100 ml
Sterilize by filtration.
Adapted and modified from: Methods in Molecular and Cellular Biology 1992,
3;15-22.
