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DNA Samples Preparation
YEAST ORF AMPLIFICATION USING
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 ---- ----
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:
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.
DNA PRECIPITATIONS => See 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.
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Preparation of Slides