Preparation of DNA Samples
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Preparation of DNA Samples
Last updated: December 3, 1999YEAST 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.) |
