Easy YAC Preparation Method

YAC TRANSFORMATION OF C. ELEGANS USING TOTAL YEAST GENOMIC DNA

[This method is described in The Worm Breeder's Gazette (1997) A. Davies and J. Shaw, 15(1)]

 

1. PREPARING YAC (+ Yeast) DNA BY CsCl PURIFICATION.

(Adapted from Stiles (1983) Methods in Enzymology 101: 296-297)

Grow 3ml culture of yeast strain carrying your YAC of interest in selective media (e.g. CM URA- TRP-) @ 30 C for 1-2 overnights.

Add culture to 50ml YPD media in flask, shake O/N 30 C.

Spin down yeast in 50 ml tube in benchtop centifuge (setting #5), 2 min, wash with 3.5ml 1M sorbitol, spin down again.

Resuspend in 3.5ml of solution containing:- 1M sorbitol, 0.1M EDTA (pH8.0), 0.1% B-Mercaptoethanol.

Add 0.3ml 2.5mg/ml zymolase or 0.3ml 10mg/ml yeast lytic enzyme (cheaper but not as effective), incubate at 37 C for at least 1 hour. [It is important to check cells before continuing. This treatment should generate spheroplasts which will burst in dH2O (check under microscope). If you have a high proportion of spheroplasts continue, if not wait longer or even add more enzyme.]

Centrifuge in benchtop centrifuge, setting#6, 3 min. Resuspend in 3.2ml TE (10mM Tris.Cl pH8.0, 1mM EDTA).

Add:- 0.32ml of 0.5M EDTA (pH8.0), 0.16ml of 2M Tris Base (pH not adjusted), 0.16ml 10% SDS, 10ul of DEPC (diethyl pyrocarbonate). Mix by inversion, incubate at 65 C for 30 min.

Add 0.8ml 5M potassium acetate, chill on ice for about 1hr.

Centrifuge 15,000g, 4 C, 10 min.

Transfer supernatant to 30ml tube, add 12 ml of room temperature ethanol.

Pellet DNA by centrifugation at 15,000g, 15 min.

Resuspend in 3.0ml TE, add 3.87g CsCl, make up to 4.9ml.

After CsCl is dissolved (by swirling), add 0.1ml ethidium bromide (10mg/ml).

[It may be useful at this point to spin down any precipitate that has formed. This can interfere with the banding process later.]

Transfer to ultracentrifuge tube (1/2 X 2 inches (13 X 51 mm) quick seal type tubes is what I have been using), balance tubes and heat seal.

Spin in vTi65 rotor at 50,000rpm (or equivalent) at 22 C for 18hr.

Using UV illumination recover DNA band using 1ml syringe and 20-21 guage needle. [Cutting off the top of the tube or inserting a needle at the top of the tube may help prevent bubbles which can disrupt your band.]

Dilute sample with 1.5 volumes of dH2O and extract ethidium bromide by adding equal volumes of isoamyl alcohol, mixing gently and removing alcohol until colour is gone.

Precipitate DNA with 2.5 volumes of ethanol at room temperature.

Centrifuge and resuspend in 50-500ul TE depending on yield (if your spheroplast step went well then 300-500ul is more appropriate).

[Much of the above method, apart from the CsCl spin, is fairly standard for preparing yeast DNA. You may find a method that is easier but given that this has worked for me I've stuck with it. The method could probably be scaled down because you should end up with much more DNA than you need but in order to see the band in the CsCl I suspect you have to start with a minimum amount.]

 

2. INJECTING WORMS

I try to keep my pRF4 (rol-6(d)) at 100ng/ul in the injection solution.

I have injected the YAC DNA (+yeast genomic) at concentrations from 15ng/ul to 150ng/ul.

In the 15-50ng/ul range I generated transformed lines at a frequency of about 1/10-1/15 of the F1 rollers I generated. From 27 transformed lines I found 3 lines that rescue.

When I injected YAC DNA (+yeast genomic) at concentrations in the range 75-150ng/ul, I achieved a similar frequency of stable transformants and found rescuing lines at frequencies from 20-50%.

So I would recommend starting at 75-100ng/ul, if you have trouble getting transformants then try a lower concentration.

Good Luck, Andrew Davies, Shaw Lab, Minnesota