Day 4 Recovery of DNA
- Spin down EtOH precipitates. Wash with 70% EtOH. Dry briefly in speed vac.
- Resuspend Pellets in 150 uL TE (equivalent to 10 uL chromatin solution per 1 uL).
- Resuspend Totals and Sups in TE volume equivalent to 1 mL chromatin solution per 1 uL (e.g. 500 uL TE for Total corresponding to 0.5 uL chromatin solution).
PCR
I use 3 mL of each sample to program a 50 uL PCR reaction. (Note: Given the volumes used to resuspend various samples, the chromatin solution equivalent for Total or Sup is 1/10 that used for Pellet). Controls include no DNA and plasmid or good genomic DNA as a positive control.
| Reaction Conditions: | DNA (3 uL) | Program: 95℃, 3 minutes |
| (in 50 pL) | lx Taq Bfr | |
| 1.5 mM MgCl2 | 95℃, 30 seconds | |
| 0.6 uM each primer | Tm-5℃, 45 seconds | |
| 0.2 mM dNTPs | 72℃, 60 seconds | |
| 0.5 uL Taq | Amplify for 24 cycles, total. | |
| 72℃, 5 minutes | ||
| 4℃ |
Add 5x Bluejuice. Analyze l/3 to 1/2 reaction on 2.5% agarose gel or 8% PAGE. Remember to be quantitative!! Photograph gel with 55 film and scan in negative to "quantitate."
Slot Blot
Dilute aliquot of each sample in 6xSSC. Denature at 100℃ ~10 minutes. Place immediately on ice. Apply samples to Nytran. Wash 2x with 6xSSC. UV crossTink filter prior to hybridization.
Southern Blot
Usually need to digest at least 1/2 of Pellet samples to see a signal; therefore, aliquot 75 mL to new Eppendorf tube and reduce volume in speed vac. Alternatively, if Southern blot analysis is anticipated, resuspend Pellets in a smaller volume initially. For Totals and Sups, digest an amount of chromatin solution equivalent to 1/5 to 1/10 amount used for Pellets.
