YAC library - YAC DNA isolation procedures

Thursday, December 04, 2003

Description
The library is a L. esculentum library of VFNT Cherry and Rio Grande-PtoR. The cosmid library was made from EcoRI partially digested total genomic DNA cloned into the BamHI site of pYAC4, which was size fractionated on a CHEF gel and selected for a range of approx. 150-200 Kb. Average insert size of the library ranges from 50 to 400 Kb

Procedure
YAC DNA isolation procedures

Total DNA-Prep
This procedure is for the isolation medium size DNA segments, and it is suitable for PCR and Southern hybridization.

Grow yeast cells from a single red colony in 50 ml AHC (or other minimal media such as SD) at 30 C for about 36 to 48 h to an OD of A260 about 2.5.
Centrifuge 5 min at 4-5000 X g .
Wash pellet with st dd H20.
Centrifuge 5 min at 4-5000 X g .
Suspend in 5 ml CBS + 25 ul YLE, incubate for 1 hr* at RT.
- CBS may be replaced by SCE. Instead of YLE (which is a stock solution) 2 mg Lyticaze may be used.
- up to this stage cells could be pipette vigorously.

*1 hr incubation may be too long. Measure OD at A 800 every 10 min (1:20 dilution in H20, in 1 ml cuvets 950 ul H20 + 50 ul of cell solution). Stop incubation at 20-30% of the original OD.

Pellet the spheroplasts 5 min at 1000 rpm.
Suspend the pellet in 5 ml TE+ + 500 ul 10% SDS; incubate 30 min at 65 C.
Transfer supernatant to new tubes, add 1.5 ml 5 M K- Acetate. Incubate 1 hr on Ice.
Centrifuge for 10' at 12,000 X g and 4 C.
Transfer supernatant to new tubes, add 1 Vol. isopropanol (RT).
Centrifuge for 10 min at 12000 X g
Suspend wet pellet in 3 ml TE (1M Tris 1M EDTA pH 8.0).
Centrifuge for 10 min at 12000 X g
Transfer supernatant to new tubes, add 150 ul RNAase A (1 mg/ml), and incubate at 37 C.
Add 0.1 Vol. 3M Na-Acetate + 0.5 Vol. isopropanol.
Centrifuge for 30 sec at 12000 X g.
Suspend in 400 ul TE
YLE: 20,000 U Yeast Lytic Enzyme (ICN # 152270 or Sigma L-5263)/ ml CSB II)

CSB I: 1 M Sorbitol
0.1 M Na-Citrat pH 5.8
10 mM EDTA
30 mM -ME (not necessary)

CSB II: 10 mM Tris-HCl, pH 7.2
20 mM NaCl
50 mM EDTA

TE+: 50 mM Tris-HCl, pH 7.4
20 mM EDTA

Superpools DNA preparation
A rapid procedure appropriate for the preparation of superpools DNA for screening by PCR.

For each YAC microtiter plate, prepare a 14-15 cm Perti dish with YPD agar medium.

Using the plate replica device stamp each YAC microtiter plate on a Perti dish and grow for 48 h at 30 C.

Scratch the 96 colonies from the plate into 10 ml TE with 20% Glycerol. Take 1 ml in 2.0 ml tubes for DNA prep. Store the rest at -80 C for further use.

Add 250 ul glass beads (0.45-0.50 mm, autoclaved in 2.0 ml tubes)

Add 400 ul Extraction buffer

1X Extraction buffer:
For 200 ml
200 mM Tris HCl pH 7.5 80 ml 0.5 M Tris-HCl pH 7.5
250 mM NaCl 10 ml 5.0 M NaCl
25 mM EDTA 25 ml 200 mM EDTA pH 7.0 0.5% SDS
Total 195 ml- autoclave add 5 ml 20% SDS

Vortex cells + glass beads + buffer for 5 min

Transfer supernatant to new tubes

Add cold Isopropanol, mix for 2 min at RT.

Centrifuge 5 min at 13000 rpm, and discard supernatant.

Suspend pellet in 400 ul H20, and vortex.

For 50 ul PCR use 1 ul

Yeast Chromosomes and Large Scale YAC-Isolation (Carle and Olson, 1985)
Grow yeast cells from a single colony in 50-100 ml YPD. Divide into 50 ml tubes
Harvest cells at 4 deg C (not essential) centrifuge (5 min, 5000 rpm, 2000 rpm or less is also OK). Re-suspend the pellet in 50 ml of 50 mM EDTA pH 7.5 in a single bottle and repeat the centrifugation step.

Yeast Storage Medium (Glycerol stock)
1% Bacto Yeast Extract
2% Bacto Peptone
2% Dextrose
20% Glycerin

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YAC library - YAC DNA isolation procedures