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| Preparation of DNA Samples
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| [ 文章来源： | 文章作者：
| 发布时间：2006-10-05|
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Preparation of DNA SamplesLast updated: December 3, 1999
YEAST ORF AMPLIFICATION USING
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| One rxn
| One 96-well plate
(100 rxns)
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| RESGEN SPECIFIC PRIMERS:
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| Program: 92C (30"), 56C (45"), 72C (3'30") / 36 cycles
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| Forward primer (20 uM)
| 5
| -
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| Reverse primer (20 uM)
| 5
| -
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| 10X PCR buffer (Perkin Elmer)
| 10
| 1000
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| MgCl2 (25 mM)
| 8
| 800
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| 100X dNTPs (25 mM each)
| 1
| 100
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| Yeast genomic DNA (0.2 ug/uL)
| 0.2
| 20
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| ddH2O
| 70.45
| 7045
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| AmpliTaq (5 U/uL)
| 0.35
| 35
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| ----
| ----
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| 100 uL
| 88 uL aliquots
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| .
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| RESGEN UNIVERSAL PRIMERS
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| Program: 92C (30"), 56C (45"), 72C (3'30") / 25 cycles
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| Forward primer (30 uM)
| 3
| 300
| 5' gga att cca gct gac cac c 3'
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| Reverse primer (30 uM)
| 3
| 300
| 5' gat ccc cgg gaa ttg cca tg 3'
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| 10X PCR buffer (Perkin Elmer)
| 10
| 1000
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| MgCl2 (25 mM)
| 8
| 800
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| 100X dNTPs (25 mM each)
| 1
| 100
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| Yeast ORF DNA (0.5 ng/uL)
| 4
| -
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| ddH2O
| 73.6
| 7360
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| AmpliTaq (5 U/uL)
| 0.4
| 40
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| ----
| ----
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| 100 uL
| 88 uL aliquots |
GEL ELECTROPHORESIS OF PCR REACTIONS (quality control)
| Agarose gel:
| 1% agarose, 1X TAE, 0.5 mg/mL ethidium bromide
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| Buffer:
| 1X TAE, 0.5 mg/mL ethidium bromide
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| Loading dye (6X):
| 15% Ficoll-400, 0.25% xylene cyanol FF, 0.25% bromophenol blue
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| 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).
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| 2.
| Pour gel with four combs of 26 wells each.
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| 3.
| Add 1 uL 6X loading dye to PCR reactions in PCR plates.
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| 4.
| Load 6 uL DNA size ladder in lane #1 of each row.
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| 5.
| Using a 12-channel pipettor, load samples A1-A12 into alternating lanes 2, 4,..., 24.
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| 6.
| Load samples B1-B12 into alternating lanes 3, 5,..., 25.
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| 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.
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| 8.
| Run at 70-80V until the first dye band (XC FF) is halfway to the next row of wells.
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| 9.
| Take a high (~1") and low (~6/30") exposure photographs.
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| Compare to predicted ORF sizes and for the presence of significant doublets.
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| 10.
| Repeat PCR rxns for failed ORFs. NOTE:
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| For 2nd PCR attempt, sort failures by gene size, doublets, etc., and modify reaction conditions accordingly.
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| For genes that still give PCR failures, design new primers, e.g. to amplify subregions of genes. |
DNA PRECIPITATIONS => See
| 1.
| Transfer PCR reactions to 96-well U-bottom tissue culture plates (Costar #3790).
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| Transfer 3 uL back to PCR plates for check gels (see above).
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| 2.
| Dry down volume in U-bottom plates to ~50 uL. (High temp. speec vacuum for 1 hr for 8 plates.)
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| The drying will be uneven, with wells around the edges experiencing more evaporation. 1 hr gets all the wells down to ~50 uL.
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| 3.
| Add 1/10 vol. 3M sodium acetate (pH 5.2) + 2.5 volumes ethanol.
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| Store at -20C for a few hours to overnight.
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| 4.
| Centrifuge in Sorvall RC-3B at 3500 rpm for 1 hr (H-6000A rotor, RCF = 3565 g).
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| 5.
| Remove supernatant with 12-channel aspirator (Wheaton/PGC Scientifics #851388).
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| 6.
| Add 100 uL of ice-cold 70% ethanol and centrifuge again for 30 min.
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| 7.
| Dry the pellets in speec-vac for 10 min.
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| 8.
| Resuspend DNA in 100 uL dH2O overnight.
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| 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.
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| 10.
| Dry down print plate sets in speed vac.
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| Tightly seal plates with aluminum foil (R.S. Hughes #425-3) for long-term storage at room temperature.
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| 11.
| Before use, resuspend one print set in 4 uL 3XSSC overnight.
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| 12.
| Spot DNA onto polylysine slides with 16-tip or 32-tip arrayer.
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| Dry down used print plates for storage until next use. (One set of print plates can be used multiple times.) |
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Preparation of 