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2004-04-30 10:51:42 | Hit : 4757 | Vote : 1222

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Full Scale YAC PLUG PREP PROTOCOL

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Full Scale YAC PLUG PREP PROTOCOL
Bob Waterston
1. Streak YAC from glycerol stock onto an AHC plate.
2. Inoculate from a single colony, or from a glycerol stock that was made from a single
colony, into 250 ml AHC in 1 liter flask. Shake at 250 rpm at 30¡ C for 40-48 hrs.
3. Before collecting cells by centrifugation. Obtain an optical density of the growths. The
spectronic should be set at 600 nm and the mode should be set for absorbance. Place 3.9
ml of ddH20 into the cuvette. Then place .1ml of cells into the cuvette. The absorbance
should be within the parameters of the 0/D chart below. The chart will have volumes
corresponding to the O/D's. The chart is as follows:
absorbance Sol. I + .1% BME 2 % seaplaque in Sol. I
-------------- ---------------------- ----------------------------
0.10-0.12 add 1.7 ml add 4.0 ml
0.12-0.14 add 2.0 ml add 4.5 ml
0.14-0.16 add 2.2 ml add 5.0 ml
0.16-0.18 add 2.4 ml add 5.5 ml
0.18-0.20 add 2.6 ml add 6.0 ml
0.20-0.22 add 2.9 ml add 6.7 ml
0.22-0.25 add 3.2 ml add 7.5 ml
3a. Melt 2% Sea Plaque GTG agarose in sol. I and place at 50o.
4. Collect cells by centrifugation (3k rpm x 5 min.) in 250 ml bottles, drain well,
resuspend gently in 50 ml of ice cold 50mM EDTA pH 8.0 . Transfer to a 50 ml tube and
repeat spin at (3k rpm x 10 min.). Drain, yield should be about 3 grams wet wt. Repeat
wash with ice cold 50mM EDTA.
5. Resuspend cells in freshly made solution I + 0.1% BME, and vortex gently.
6. To create plug, place cells at 50o C. Add 250 ul of ~ 5 mg/ml zymolyase (100T) in
water prepared 1/2 hour ahead of time, mix let then add 2% Sea Plaque GTG agarose in
solution I, equilibrated at 50o C. Mix by pipetting gently and transfer entire contents
immediately to plug molds. Pipette only 300 ul into each plug form. Allow to set on a flat
surface for five minutes.
7. Chill at 4o C for 10 min.
8. Place plugs into purple boxes. Cover with about 50 ml of sol. I + 0.1% BME + 20 mg
zymolyase / 50ml of solution.
9. Incubate at 37o overnight with gentle shaking ( 35 rpm). 10. Remove solution and for
all samples and add 50 ml of Sol. III + 20 mg/50ml pK to the box. Incubate the plugs at
50o C overnight.
11. Load plugs onto first gel 1% seakem 180ml. Put remaining plugs through the rest of
the protocol.
12. Remove solution and for all samples add 100 ml of Sol. III to each purple box.
Incubate at 37o C overnight.
12. Remove the solution III for all samples and add 100 ml of .5 M EDTA to purple
boxes. Incubate at 37o C overnight.
13. Remove the solution for all samples add 100 ml of .5 M EDTA to purple box.
Incubate at 37o C overnight.
14. Store plugs at 4o C in .05M EDTA.
Solution I
1m sorbitol/20 mM EDTA pH 8.0
Solution II
450mM EDTA/10 mM Tris (pH 8.0) + 01% BME
Solution III
1% lithium dodecyl sulfate/500mM EDTA/10 mM Tris (pH 8.0)
GSC Production Protocols
http://genome.wustl.edu/gsc/GSC_Protocols/footer.htm [8/29/2001 10:25:18 AM]