Zebrafish YAC DNA

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The zebrafish YAC clones distributed by Research Genetics, Inc. are provided as stab cultures in YPD agar. Glycerol stocks of each clone should be made as soon as possible. The clone will remain viable as a stab culture up to one week if stored at 4 oC

Average Insert size: 480 kb
DNA source: 5 day embryo
Vector, Host Strain: pRMLl and pRML2. Saccharomyces cerevisiae J57D

J57D genotype: MATot leu2-3,-1 12 ura3-52 trpl his3-2,-15 ade2 can1

Cloning site: EcoRI

Glycerol stock preparation

1. Use a sterile loop to innoculate a tissue culture flask (Falcon tube) containing 15 m1 YPD medium with the clone stab.

2. Incubate at 30 oC with shaking overnight or until turbid.

3. Add 4 ml of sterile 80% glycerol. Mix thoroughly

4. Dispense into microtube and freeze at ―80 oC or less.

5. Revive the yeast by transferring a small portion of the frozen sample onto a YPD agar plate or directly into YPD.

YPD medium:

1% Bacto-yeast extract
2% Bacto-peptone
2% Glucose

Reference

1. F. Spencer, G. Ketner, C. Connelly, and P. Hieter. Targeted recombination-based cloning and manipulation of large DNA segments in yeast. Methods: a companion to Methods in Enzymology 5: 161-175 (1993).

　

DNA preparation:

(From Hotfman and Winston Gene 57: 267-272 [1987], modified by AK.)

1. Grow small cultures (15 ml) overnight at 30 oC in 15 ml YPD to saturation. (Care should be taken to innoculate).

2. Centrifuge at 1500 rpm for 5min. Decant the supernatant. .

3. Add 0.5 ml H2O. Suspend and transfer to microtube. Spin again for 5 sec at 14000 rpm.

4. Decant the supenatant and briefly vortex the tube to rcsuspend the pellet in the residual liquid.

5. Add 0.2 ml of 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl (pH 8), 1 mM EDTA. Add 0.2 ml of phenol:chloroform:isoamyl alcohol (25:24..1). Add 0.3 g of acid-washed glass beads (0.45-0.5 mm, Sigma).

6. Vortex for 3-4 minutes and add 0.2 ml TE.

7. Centrifuge for 5 minutes in a microfuge

8. Take upper phase (There is a hard interphase). Add 1 ml ethanol (RT), mix well by inverting tube. Immediately centrifuge for 2 min.

9. Dissolve ppt in 400 ul TE,

Add 3 ul of 10 mg/ml RNase A. 37 oC for 20 min.

Extract with phenol choroform

Add 10 ul of 4 M ammonium acetate plus 1 ml of l00% ethanol. Mix by inversion.

10. Centrifuge for 2 minutes in a microfuge. This is〜2-4 ug of DNA. Dry and dissolve the ppt in 50 ul of 0.1 x TE, and use 10-20 ul for DNA digestion of plasmid rescue.