Here are the responses to my recent request for a good DNA mini prep for
Chlamydomonas.
Hank Latorella
Biology Dept.
SUNY Geneseo
Geneseo, NY 144454
latorell at uno.cc.geneseo.edu
********************************************************
RESPONSES TO DNA MINI PREP REQUEST
Try Scott Newman's method, described in Genetics 126:875. I've
found it quick & reliable. No problems with polysaccharides that I
know of.
Peter Luykx, Dept. of Biology, Univ. of Miami.
PLUYKX at umiami.ir.miami.edu
***************************************************************
The following is a general mini-prep protocol we have been using to
extract both RNA and DNA from green micro-algae (ncluding lots of
Chlamys). If youare not interested in the RNA, you may want to opt
for a protocol that eliminates the RNA with RNAase.
1. Harvest cells or filaments
a. unicells by centrifugation in sterile centrifuge tubes
2. Wash cells in 50mM Tris HCl (pH 9.0)
a. discard wash
3. Resuspend and break material in 500 ul lysing buffer*
a. unicells with
i. microfuge "mortar and pestle"
ii. ultrasonic probe (10-20 pulses)
c. check cell breakage by microscopy
4. Add 50 ul Guanidine HCl (8 M) to broken cell extract (precipitates
polysaccharide and SDS)
a. vigorously mix for 1 minute
5. Add equal volume (550 ul) of 49:1 in fume hood
a. mix for 2 minutes
b. spin (at max.) for 2 minutes
6. Recover aqueous phase
7. Add equal volume (550 ul) of Amresco 25:24:1 in fume hood
a. mix for 2 minutes
b. spin (at max.) for 2 minutes
8. Recover aqueous phase
9. Add equal volume (550 ul) of 49:1 in fume hood
a. mix for 2 minutes
b. spin (at max.) for 2 minutes
10. Recover aqueous phase
11. Add 1 volume isopropyl alcohol (2-propanol)
12. Freeze for at least 2 hours or overnight
13. Pellet nucleic acid by centrifugation
a. 10-20 minutes (at max. speed)
14. Discard supernatant
15. Dry pellet in DNA SpeedVac (5-15 minutes at medium; caps
open)
16. Dissolve pellet in 200 ul water (ART tips)
17. Add equal volume (200 ul) LiCl 4 M (ART tips)
18. Refrigerate 4-5 hours or overnight
19. Pellet RNA by centrifugation (label tube as RNA)
a. 10-20 minutes (at max. speed)
20. Transfer supernatant (with DNA) to new microfuge tube (label
tube as DNA)
RNA:
21r. Dissolve RNA pellet in 200 ul water
22r. Sample ready for UV spec
DNA:
21d. Add 2 volumes (800 ul) ethanol to DNA
22d. Freeze for at least 2 hours or overnight
23d. Pellet DNA by centrifugation (10-20 min @ max)
24d. Discard supernatant
25d. Dry pellet in DNA SpeedVac; caps open
26d. Resuspend pellet in 200 ul water (ART tips)
27d. Sample ready for UV spectrophotometry
28d. Add 2 volumes (ca. 400 ul) ethanol to remaining nucleic acid
preps
29d. Freeze for at least 2 hours or overnight
30d. Pellet nucleic acid by centrifugation
31d. Discard supernatant
32d. Dry pellet in DNA SpeedVac; caps open
33d. Resuspend nucleic to standard volume (1ug/ul) in water
(ART tips)
*Su and Gibor, 1988, Anal. Biochem. 174:650-657
biol_mab at vax1.utulsa.edu (Mark Buchheim)
***********************************************************************
Chlamydomonas Mini Whole Cell DNA Prep.
Originally developed by Scott Newman in Boynton-Gillham lab.
I'm told they now grow 1 ml aliquots of cells in multiwell plates
rather than working from patches on agar.
-Scrape cells off plate into 1.5 ml Eppendorf tube that contains 0.5 ml
TEN buffer.
-Resuspend vigorously by vortexing, spin for 10 sec. and aspirate off
supernatant.
-Resuspend cells in 150 ul H2O on ice and add 300 of SDS-EB buffer,
vortex
to mix.
-Extract once with 350 ul phenol/CIA(1:1) for few min by vortexing,
separate phases by centrifugation for 5 min., transfer aq. phase to a
new tube.
-Extract once with 300 ul CIA (24:1), transfer aq. to a new tube.
-Add 2 volumes abs. ethanol, incubate on ice for 30 min., centrifuge
for 10 min., wash pellet once with 200 ul 70% ethanol.
-Dry pellet and resuspend in ca. 40 ul H2O, for Southern analysis use
about 1-3 ul.
TEN=10 mM Tris-Hcl, 10 mM EDTA, 150 mM NaCl.
SDS-EB= 2% SDS, 400 mM NaCl, 40 mM EDTA, 100 mM Tris-Hcl, pH
8.0.
Elizabeth Harris
chlamy at acpub.duke.edu
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Here's a protocol I came up with a year or so ago to quickly make up
enough genomic DNA for PCR reactions. since then, the Chlamy
Newsletter has published a similar miniprep protocol.
1) Resuspend one to several loopfuls of Chlamydomonas in 100 ml of
lysis buffer (10mM Tris pH 8.0, 1mM EDTA, 3% SDS) in a microfuge
tube, or pellet 1ml of liquid culture 30" in a microfuge. Remove the
supernatant by aspiration and resuspend the pellet in 100 ml lysis
buffer.
2) Incubate 15' at room temperature, mixing occasionally.
3) Add 500 ml TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) in order to
help separate phases in subsequent extractions. Add 1/10 volume
3M NaOAc, pH 5.2.
4) Phenol extract once. Chloroform extract twice.
5) Add 1.1 volumes isopropanol. Pellet 15' in microfuge. Wash with
70% ethanol and air dry. Resuspend in 50 ml TE. A typical yield is 1
- 2 ug of genomic DNA. I use about 5 ul in a PCR reaction.
****************
More recently, I've tried other methods, including:
1) Resuspend a couple of healthy loopfuls of cells in SDS extraction
buffer (from Weeks et al. Anal. Biochem. 152:376-385;1986): 2%
SDS, 400mM Nacl, 40mM EDTA, 100mM Tris-HCl, pH 8.0. Mix
thoroughly, but try not to introduce too many bubbles. The volume
used depends on the amount of cells used.
Incubate 15' @ RT or 65 C (depending on how clumpy the cells are).
2) Phenol extract. If the aqueous phase is cloudy, add a couple
hundred microliters of water to the tube and spin again. Chloroform
extract.
3) There's enough salt in the aqueous phase to isopropanol
precipitate directly.
I get more than a few micrograms of digestable DNA from this
protocol, but I haven't tried it in a PCR reaction.
I've only had problems with polysaccharides when doing CsCl
preps. Even then, when I've had starch in my preps, a quick spin in a
microfuge gets it out of the way after the prep is resuspended.
Hope this helps.
Tony
Anthony Palombella <palomb at beagle.Colorado.EDU>
***********************************************************************
You may use our protocol to isolate Chlamydomonas DNA from small
cultures.
Take 2 ml of a very dense grown culture and spin it down in 2 ml
Eppendorff tubes at 3000 rpm.
Resuspend the pellet in 500 ul of CTAB-Buffer.
2% (w/v) CTAB
100 mM Tris-HCl, pH 8
1,4 M NaCl
20 mM EDTA (pH 8)
2 % (v/v) beta-Mercaptoethanol
Incubate the solution at 65 degree C for 1 h.
Extract with 500 ul of Phenol/Chloroform/Isoamylalcohol (25:24:1).
Take the upper phase and pellet it with 0.7 volumes of isopropanol
for 15 min at 4 degree C. Spin down at 12000 rpm for 20 min.Wash
the pellet
Good luck
Gernot
--
Gernot Gloeckner,203-2766 <gloeckne at sun1.ruf.uni-freiburg.de>
*********************************************************
In response to the request for a miniprep protocol for Chlamy DNA, I
have some preliminary information that might help. There was a
paper published in Biotechniques recently (Goodwin and Lee, 1993,
15:438-444) "Microwave Miniprep of Total Genomic DNA from Fungi,
Plants, Protists, and Animals for PCR".
A temporary technician in our lab tried this for DNA isolation from a
green alga Monoraphidium. This is a very tough, tiny alga from
which it is very difficult to isolate unsheared, clean DNA. In a
preliminary experiment using the microwave technique, she was
able to isolate about a microgram of digestable DNA from 40 ml of a
medium density culture. Unfortunately, she left the lab and we
haven't had a chance to pursue this further, but it looked promising.
Granted, its not as "mini" a prep as the one posted by Dr. Harris, but
it may be more useful for cells that are more difficult to lyse than
Chlamy.
Terri Dunahay
National Renewable Energy Lab
dunahayt at tcplink.nrel.gov
**********************************************************
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
usingtable-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
5 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 -20C.
Concentration of DNA is 2-5g/ 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 12.5mM
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
700mM
Water to 100 ml
Sterilize by filtration.
Adapted and modified from: Methods in Molecular and Cellular
Biology 1992, 3;15-22.
surzycki at sunflower.bio.indiana.edu
A.H. LATORELLA
BIOLOGY DEPARTMENT
SUNY GENESEO
GENESEO, NY 14454
(716) 245-5312
LATORELL at UNO.CC.GENESEO.EDU
