Overgo Probing of High-Density Filters
(John D. McPherson) Washington University, St. Louis, Missouri
Contents
- A diagramatic overview
- Protocol
- Hybridization solution
- Overlapping oligo labelling
- Preparation of OLB(-A,-C,-N6)
I have been using double-stranded 36mers form STS to probe high-density PAC/BAC filters. The single-stranded overhangs (14 bases) are filled in with 32P labelled ATP and CTP. POsitive clones are then fingerprinted by complete restriction enzyme digestion and contigs assembled.
Hybridizations:
Note: Done in a hybridization oven at 58°C. This temperature works well for overgos with GC content between 40% and 60%. Using overgos which are AT-rich may require lowering the hybridization temperature.
- 50 ml of warmed hyb solution is added to a 30 cm x 4 cm hyb bottle. Filters are prewet with warmed 2xSSC and then each filter is rolled and inserted into the bottle. With the cap on, the bottle is rotated to allow the filter to unroll slowly and not trap air bubbles. Each filter is added separately at this time to ensure that each is prehyb'd adequately. Routinely, 7 filters are placed in the same bottle. Make sure that all filters are rolled in the same direction and that the bottle is set to about 2/3rds maximal.
Filters are prehyb'd for 4 hours the first time they are used and 1-2 hours thereafter. I have not been adding any sheared salmon testes DNA.
- After prehyb'ing, 10 ml of the hyb is removed from each bottle and combined, the labeled oligos are denatured at 90°C for 10 minutes. They are then added and mixed by pipetting. 10 ml is then added to each bottle to transfer the probe. Probes are allowed to hybridize overnight typically; however, 2 day hybridizations give somewhat stronger signals. This may be useful with older filters.
Washing:
- Hyb solution is removed and the bottle filled 2/3rds with room temperature 2xSSC, 0.1% SDS. The bottle is returned to the oven and rotated for about 30 min.
- Filters are now transferred to a larger tub on a rotary platform and washed as follows:
2L 1.5xSSC, 0.1% SDS 58°C 30min
2L 0.5xSSC, 0.1% SDS 58°C 30min
Autoradiography:
- Filters are sealed in plastic bags and exposed using XAR5 film at -70°C.
- Overnight exposures are usually all that is needed. This can be increased as the filters age.
Hybridization solution I (7% SDS)
Composition For 1000 ml
1% BAS (Fraction V, Sigma) 10g
1mM EDTA 2ml 0.5M EDTA (pH8.0)
7% SDS (use 99.9% pure SDS) 70g
0.5M sodium phosphate* 500ml 1M sodium phosphate*
To make 1000ml: (use autoclaved deionized H2O)
- Heat 100ml H2O for 15s in microwave on high. Add 10g BSA and stir to dissolve.
- To 500ml 1M sodium phosphate*, add 200ml H2O, 2ml 0.5M EDTA and 70g SDS.Stir until the SDS is dissolved (1 hr or so).
- Add the dissolved BSA and make volume to 1000ml.
- Filter the hybridization solution and store at 37°C tp p[revent the SDS from precipitating.
*Note: 1M sodium phosphate
must be made as follows (this is really 1M with respect to sodium but follows the referenced nomenclature): 134g Na
2HPO
4.7H
2O, add 4 ml 85% H
3PO
4 and make to 1000ml.
- Heat stock solution (10pmol/µl) of mixed oligos at 80°C, 5min, 37°C, 10min, and store on ice.
- Labeling reaction (10µl): oligo + H2O = 5.5µl
| oligonuceotides |
|
10 pmol each (1µl in this case) |
| BSA (2mg/ml) |
|
0.5 µl |
| OLB (-A,-C,-N6) |
|
2.0 µl |
| 32P-dATP* |
|
0.5 µl |
| 32P-dCTP* |
|
0.5 µl |
| H2O |
|
to 10 µl (4.5µl in this case) |
| Klenow fragment |
|
1 µl (2U/µl) |
Inclubate ar room temperature for 1 hour.
- Remove unincorporated nucleotides using Sephadex G50 columns. Denature probes before adding to blots.
Preparation of OLB(-A,-C,-N6)
OLB(-A,-C,-N6) = A:B:C (1:2.5:1.5)
- Solution O
- 1.25M Tris-HCL, pH8
- 125mM MgCl2
- Solution A
- 1 ml solution O
- 18µl 2-mercaptoethanol
- 5µl 0.1M dTTP
- 5µl 0.1M dGTP
- Solution B
- 2M HEPES-NaOH, pH6.6
- Solution C
- 3mM Tris-HCl, pH7.4
- 0.2mM EDTA
- Aliquot and store at -20°C.
Overlaping oligo labelling
Protocol
上一篇：BAC DNA Isolation from 200 ml Cultures by a Cleared Lysate Method Followed by Do 下一篇：BAC end-sequencing
