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Preparation of DNA Samples

Preparation of DNA Samples

Last updated: December 3, 1999

YEAST ORF AMPLIFICATION USING RESGEN PRIMERS

One rxn One 96-well plate
(100 rxns)
RESGEN SPECIFIC PRIMERS: Program: 92C (30"), 56C (45"), 72C (3'30") / 36 cycles
Forward primer (20 uM) 5 -
Reverse primer (20 uM) 5 -
10X PCR buffer (Perkin Elmer) 10 1000
MgCl2 (25 mM) 8 800
100X dNTPs (25 mM each) 1 100
Yeast genomic DNA (0.2 ug/uL) 0.2 20
ddH2O 70.45 7045
AmpliTaq (5 U/uL) 0.35 35
---- ----
100 uL 88 uL aliquots
.
RESGEN UNIVERSAL PRIMERS Program: 92C (30"), 56C (45"), 72C (3'30") / 25 cycles
Forward primer (30 uM) 3 300 5' gga att cca gct gac cac c 3'
Reverse primer (30 uM) 3 300 5' gat ccc cgg gaa ttg cca tg 3'
10X PCR buffer (Perkin Elmer) 10 1000
MgCl2 (25 mM) 8 800
100X dNTPs (25 mM each) 1 100
Yeast ORF DNA (0.5 ng/uL) 4 -
ddH2O 73.6 7360
AmpliTaq (5 U/uL) 0.4 40
---- ----
100 uL 88 uL aliquots

GEL ELECTROPHORESIS OF PCR REACTIONS (quality control)

Agarose gel: 1% agarose, 1X TAE, 0.5 mg/mL ethidium bromide
Buffer: 1X TAE, 0.5 mg/mL ethidium bromide
Loading dye (6X): 15% Ficoll-400, 0.25% xylene cyanol FF, 0.25% bromophenol blue
DNA size ladder: 4 mL 1X TE buffer, 1 mL 1kb ladder, 1 mL 6X loading dye

1. Start with 3 uL of PCR reactions in PCR plates, after remainder is transferred to U-bottom plates (see next section).
2. Pour gel with four combs of 26 wells each.
3. Add 1 uL 6X loading dye to PCR reactions in PCR plates.
4. Load 6 uL DNA size ladder in lane #1 of each row.
5. Using a 12-channel pipettor, load samples A1-A12 into alternating lanes 2, 4,..., 24.
6. Load samples B1-B12 into alternating lanes 3, 5,..., 25.
7. Repeat this procedure for the remaining samples, such that two sequential rows of PCR reactions are loaded into a single row of wells in alternating lanes.
8. Run at 70-80V until the first dye band (XC FF) is halfway to the next row of wells.
9. Take a high (~1") and low (~6/30") exposure photographs.
Compare to predicted ORF sizes and for the presence of significant doublets.
10. Repeat PCR rxns for failed ORFs. NOTE:
For 2nd PCR attempt, sort failures by gene size, doublets, etc., and modify reaction conditions accordingly.
For genes that still give PCR failures, design new primers, e.g. to amplify subregions of genes.

DNA PRECIPITATIONS => See Comparison of PCR Cleanup Protocols

1. Transfer PCR reactions to 96-well U-bottom tissue culture plates (Costar #3790).
Transfer 3 uL back to PCR plates for check gels (see above).
2. Dry down volume in U-bottom plates to ~50 uL. (High temp. speec vacuum for 1 hr for 8 plates.)
The drying will be uneven, with wells around the edges experiencing more evaporation. 1 hr gets all the wells down to ~50 uL.
3. Add 1/10 vol. 3M sodium acetate (pH 5.2) + 2.5 volumes ethanol.
Store at -20C for a few hours to overnight.
4. Centrifuge in Sorvall RC-3B at 3500 rpm for 1 hr (H-6000A rotor, RCF = 3565 g).
5. Remove supernatant with 12-channel aspirator (Wheaton/PGC Scientifics #851388).
6. Add 100 uL of ice-cold 70% ethanol and centrifuge again for 30 min.
7. Dry the pellets in speec-vac for 10 min.
8. Resuspend DNA in 100 uL dH2O overnight.
9. Transfer in 10 uL aliquots to 384-well plates (USA Scientific #2802-0384 or Corning Costar #6502) to make 10 duplicate print plate sets.
10. Dry down print plate sets in speed vac.
Tightly seal plates with aluminum foil (R.S. Hughes #425-3) for long-term storage at room temperature.
11. Before use, resuspend one print set in 4 uL 3XSSC overnight.
12. Spot DNA onto polylysine slides with 16-tip or 32-tip arrayer.
Dry down used print plates for storage until next use. (One set of print plates can be used multiple times.)