hi,
I have tried preparing YAC from spheroplast, and the following protocol
is writen by me and seems work fine. But I haven't tried any rescuing
experiment to confirm those DNA are clean enough for rescue.
*Purification of Yeast DNA in Solution by standard method*
*Reagents*
*Yeast wash solution (20ml)*
0.5M EDTA pH 8
1.6ml (40mM)
2-mercaptoethanol (concentrate stock)
128?l (90mM)
H_2 O
Make up to 20ml
/Add 2-mercaptoethanol just prior to use/
* *
*SCE (25ml)*
Sorbitol
4.55g (1M)
500mM Citric acid, sodium salt (pH 5.5)
5ml (100mM)
0.5M EDTA pH 8
0.5ml (10mM)
/Adjust volume to 25ml by H_2 O, autoclave/
* *
*Spheroplast lysis buffer (25ml)*
1M Tris-HCl (pH 7.4)
1.25ml (50mM)
0.5M EDTA pH 8
1.25ml (25mM)
3M NaCl
4ml (480mM)
20% SDS
1.25ml (1%)
2-mercaptoethanol (add freshly)
5?l (3mM)
/Add 20% SDS after all reagents to avoid precipitation./
/Make up volume to 25ml by H_2 O, add 2-mercaptoethanol just prior to
use, if necessary, add RNaseA to a concentration 10/?g/ml//
* *
*Protocol*
1. Inoculate yeast into 1ml of -Ura medium. Shake O/N at 30?
250. Dilute overnight 1ml culture to 10ml -Ura medium, shake O/N at 30?
250rpm
2. Harvest yeast cell by centrifugation at 3000g at room
temperature for 10 minutes.
3. Prepare 20ml of yeast wash solution. Resuspend cells in 10ml
yeast wash solution, centrifuge at 3000g at room temperature for 10mins,
and decant supernatant.
4. Repeat step 3 once.
5. Add 2ml of SCE, 15?l of 2-mercaptoethanol and 50ml
(1000U/ml) or 20mg of lyticase / zymolase
6. Incubate stationary at 37? for 2 hours, with gentle mixing
once half an hour. You can see yeast spheroplasts clump together after
complete digestion.
7. Divide cells equally into two 1.5ml eppendorf tubes,
centrifuge at 6000rpm for 10mins and remove supernatant.
8. Add 0.5ml of spheroplast lysis buffer to each tube, disrupt
the cell aggregates by pipetting up and down (vortexing seems not as
efficient as pipetting up and down because spheroplast tends to clump
together) Incubate at 65? for 20mins, mix intermittently.
9. Add 0.25ml phenol, mix, add 0.25ml of chloroform:IAA (24:1),
mix thoroughly, centrifuge at 13000g for 5 minutes.
10. Transfer 0.5ml aqueous phase into a new microcentrifuge tube
and discard the organic phase.
11. Repeat step 9&10 once.
12. Add 0.5ml of chloroform:IAA (24:1), mix thoroughly, centrifuge
at 13000g for 5 minutes.
13. Repeat step 12 once.
14. Add an equal volume of isopropanol and mix, you should see DNA
precipitating out. Use a glass loop gently fish out the DNA
(alternatively, centrifuge at ~13000g for 15 minutes, discard
supernatant. (you may need more extensive centrifugation, DNA seems less
easily pellet down compare with standard mini-prep procedure)
15. Wash DNA pellet with 400?l 70% ethanol.
16. Dry DNA under vacuum.
Add 200?l of TE (pH8) or ddH_2 O incubate at room temperature overnigh
until all DNA is dissolved, alternatively heat at 50? to dissolve
Left lane: marker
Right lane: my YAC together with yeast genomic DNA
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