Yu:Northern solns
From OpenWetWare
(Redirected from Yu:AQUEOUS Hyb BUFFER)
Northern Solutions
1.3% Agarose Formaldehyde gel(85 mL)
- Dissolve 1.16 g of agarose in 62mL of milliQ H2O
- Cool agarose to 55-60 °C in a pre-warmed water bath
- Add 8.5 mL of 10x MOPs
- Add 14.45 mL of 37% formaldehyde in the hood
- Pour gel immediately and allow to cool of at least 30 min in the hood
20x SSC
| Final Concentration | For 1 Liter | For 4 Liters |
| 3M NaCl | 175.3 g | 701.2 g |
| 0.3M NaCitrate | 88.2 g | 352.8 g |
| milliQ H2O | to 1 L | to 4 L |
10x MOPs
| Final Concentration | For 500 mL | For 1 Liter |
| 0.4 M Morpholinopropanesulfonic (MOPS) acid (free acid) | 41.9 g MOPS | 83.8 g MOPS |
| 0.1 M Na-acetate-3 x H2O | 6.8 g | 13.6 g |
| 10 mM EDTA | 10 mL 0.5 M EDTA | 20 mL 0.5 M EDTA |
| Adjust pH to 7.2 with NaOH | ||
| Store in the Dark at 4 °C | ||
100x Denhardt's
| Final Concentration | For 100 mL | For 500 mL |
| Ficoll 400 | 2 g | 10 g |
| Polyvinylpyrrolidone MW 360000 | 2 g | 10 g |
| BSA fraction V | 2 g | 10 g |
| milliQ H2O | ||
| Store at -20 °C | ||
Hybridization Buffers
Oligo (Pre)Hybridization Buffer
| Final Concentration | For 100 mL | For 200 mL |
| 3x SSC | 15 mL of 20x SSC | 30 mL of 20x SSC |
| 0.5% SDS | 5 mL of 10% SDS | 10 mL of 10% SDS |
| 1x Denhardt's | 2 mL of 50x Denhardt's | 4 mL of 50x Denhardt's |
| 100 μg/mL ssDNA | 1 mL 10 mg/mL ssDNA | 2 mL 10 mg/mL ssDNA |
| milliQ H2 | to 100 mL | to 200 mL |
Hyb Buffer
| Final Concentration | For 50 mL | For 100 mL |
| PIPES | 2.5 mL of 1M PIPES pH 6.5 | 5 mL of 1M PIPES pH 6.5 |
| Sodium Phosphate Buffer | 2.5 mL of 1M NaPO4 Buffer pH 7.0 | 5 mL of 1M NaPO4 Buffer pH 7.0 |
| Sodium Chloride | 1 mL of 5M NaCl | 2 mL of 1 mL of 5M NaCl |
| SDS | 12.5 mL of 20% SDS | 25 mL of 20% SDS |
| EDTA | 250 μL of 0.5M EDTA pH 8.0 | 500 μL of 0.5M EDTA pH 8.0 |
| ssDNA | 300 μL 10 mg/mL ssDNA | 600 μL 10 mg/mL ssDNA |
| milliQ H2 | to 50 mL | to 100 mL |
Oligo Wash Buffer
| Final Concentration | For 500 mL | For 1 L |
| 2x SSC | 50 mL of 20x SSC | 100 mL of 20x SSC |
| 0.1% SDS | 5 mL of 10% SDS | 10 mL of 10% SDS |
| milliQ H2 | to 500 mL | to 1 L |
Loading Buffers
MOPs Loading Buffer
| Final Concentration | For 1 mL | For 10 mL |
| 1x MOPs | 100 μL | 1 mL |
| 18.5% Formaldehyde | 185 μL | 1.85 mL |
| 50% Formamide | 500 μL | 5 mL |
| 4% Ficoll 400 | 40 mg | 400 mg |
| milliQ H2 | 215 μL | 2.15 mL |
| pinch of Bromophenol Blue | ||
| Store in the dark at -20 °C | ||
Formamide Loading Buffer
| Final Concentration | For 1 mL | For 10 mL |
| >99% Formamide | 0.96 mL of deionized Formamide | 9.6 mL of deionized Formamide |
| 20 mM EDTA | 4 μL of 0.5M EDTA | 400 μL of 0.5M EDTA |
| Pinch of Xylene Cyanol and Bromophenol Blue | ||
| Store in the dark at -20 °C | ||
