Tuesday, November 18, 2003

Description
Vectorette PCR of Yeast DNA

Procedure
1) Cut 1-3 g of clean DNA overnight with 8-10U of blunt cutting enzyme in 20l
Most problems come from dirty, uncut DNA. Phenol/glass bead/RNase prepared DNA works well

RsaI, AluI and DraI provide good results.

2) Heat inactivate enzyme

3) Add:

3l 10x NEBuffer used in digest
1l annealed anchor bubble
1l (400U) ligase
0.5l of 5mM ATP (50M ATP final)
25.5l Water
4) Incubate at 16 C for 9-24 hours.
5) Use 5l in 100l PCR. Perkin Elmer Ampliwax is recommended for hot start.

5 l of ligation
2.5 l of 20M specific primer [M13(-47) for mTn3 library]
2.5 l of 20M 224 primer
8 l of 2.5 mM dNTPs
10 l of Taq PCR buffer
71l Water
1l Taq DNA polymerase (5U)
Transfer to Perkin Elmer 9600 Thermal Cycler
Denature 92C, 2 minutes
35 Cycles [92C, 20sec; 67C, 30sec; 72C, 45-180sec (>1 min/1 kb)]
72C, 90sec
6) Gel purify 80 l of PCR product in 1-3% SeaKem GTG, extract with Qiaex (Qiagen), elute with 12 l of ddWater
7) Sequence 7 l with Sequenase kit from Amersham.

Use 1 l of 200-600M specific primer [M13(-47) for mTn3]. Undiluted 10 OD synthesis from Genset works well.

Use high specific activity S-35 (>1000 Ci/mmol, Amersham AG1000)

Boil 10' and fast cool in ice water.



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Anchor Bubble primers
3' GAGAGGGAAGAGAGCAGGCAAGGAATGGAAGCTGTCTGTCGCAGGAGAGGAAG 5'
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5' GACTCTCCCTTCTCGAATCGTAACCGTTCGTACGAGAATCGCTGTCCTCTCCTTC 3'


PRIMER 224 5' CGAATCGTAACCGTTCGTACGAGAATCGCT 3'



To anneal bubble primers, heat a 2-4M (in ddWater) to 65 C for 5 minutes, then add MgCl2 to 1-2 mM and allow to cool to room temperature.

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