Minipreps of DNA from Chlamydomonas

Try Scott Newman's method, described in Genetics 126:875. I've found it quick & reliable. No problems with polysaccharides that I know of.

Here is Scott Newman's protocol, as currently followed in the Boynton-Gillham laboratory. Cells can be grown as patches on agar plates, or as 1 ml aliquots of cells in multiwell plates.

• 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 ul 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.

SDS-EB = 2% SDS, 400 mM NaCl, 40 mM EDTA, 100 mM Tris-HCl, pH 8.0.

The following is a general mini-prep protocol we have been using to extract both RNA and DNA from green micro-algae (including lots of Chlamys). If you are not interested in the RNA, you may want to opt for a protocol that eliminates the RNA with RNAase.

• 1. Harvest cells by centrifugation in sterile centrifuge tubes

• 2. Wash cells in 50mM Tris HCl (pH 9.0). Discard wash.

• 3. Resuspend and break material in 500 ul lysing buffer (Su and Gibor, 1988, Anal. Biochem. 174:650-657) with ultrasonic probe (10-20 pulses). Check cell breakage by microscopy

• 4. Add 50 ul Guanidine HCl (8 M) to broken cell extract (precipitates polysaccharide and SDS). Vigorously mix for 1 minute

• 5. Add equal volume (550 ul) of 49:1 in fume hood
  • mix for 2 minutes
  • spin (at max.) for 2 minutes

• 6. Recover aqueous phase

• 7. Add equal volume (550 ul) of Amresco 25:24:1 in fume hood
  • mix for 2 minutes
  • spin (at max.) for 2 minutes

• 8. Recover aqueous phase

• 9. Add equal volume (550 ul) of 49:1 in fume hood
  • mix for 2 minutes
  • 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 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) 10-20 minutes (at max. speed)

• 20. Transfer supernatant (with DNA) to new microfuge tube (label tube as DNA)

• 21r. Dissolve RNA pellet in 200 ul water

• 22r. Sample ready for UV spec

• 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 speed)

• 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 (1 ug/ul) in water (ART tips)

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.

• 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'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

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

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 Gloeckner

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 devised by Scott Newman, but it may be more useful for cells that are more difficult to lyse than Chlamy.

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 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 ul of TE buffer (or water). Transfer the DNA solution to microfuge 1.5 ml tube.

9. Add 150 ul of 7.5 M ammonium acetate. Mix well by inverting several times.

10. Add 1000 ul of 95% ethanol and mix well.

11. Centrifuge for 10 minutes in microcentrifuge. Discard supernatant.

12. Wash pellet twice with 700 ul 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 ul of TE buffer. Add 10 ul of DNase free RNase A and 1 ul of RNase T1. Mix well and incubate for 30 minutes at 37 C.

14. Add 150 ul 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 ul 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 ul cold 70% ethanol. Remove last drop of ethanol as described above after last wash.

18. Resuspend pellet in 20-50 ul of sterilized water. Store in -20C. Concentration of DNA is 2-5 ug/ul. 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

Adapted and modified from: Methods in Molecular and Cellular Biology 1992, 3;15-22.

Stefan Surzycki

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