Steve Hahn. last modified Sat, Oct 17, 1998
Mix the following in an 0.5 ml eppendorf tube:
10-20 micrograms total yeast RNA
10 microliters hybridization mix
H2O to bring the final volume to 25 microliters
40 microliters mineral oil
Heat the reactions at 100 degrees for 2 min and transfer to 55 degrees overnight (12-16 hr) for hybridization (the exact temperature will depend on the length and composition of oligonucleotide)
After the overnight incubation, spin tubes and remove aqueous layer to 1.5 ml microfuge tubes. Add 275 microliters S1 mix and incubate at 37 degrees for 10-30 min. depending on the probe.
Add 6 microliters stop buffer 900 microliters ETOH and perform a standard precipitation. Wash pellets with 80% ETOH. Resuspend in 6 microliters of Formamide sequencing gel loading buffer containing 0.1% SDS. Load samples to an 8% sequencing gel.
Important controls to have are: (i) kinased probe with no nucleic acid, and (ii) reactions containing variable amounts of total RNA. The signal should be linear with increasing amount of RNA if the results are quantitative, (iii) load 500-1000 cpm of probe with no S1 treatment on the sequencing gel to observe the quality and size of probe with no S1 treatment.
Hybridization Mix:
5 microliters 4 M NaCl
2.5 microliters 1 M PIPES pH 6.9
1 microliter 0.25 M EDTA
1-2 x 105 cpm kinased oligonucleotide probe
H2O for a final volume of 10 microliters/reaction
| 1X S1 Buffer: |
|
10 ml 10X S1 Buffer |
| 4 mM ZnSO4 |
|
0.4 ml 1 M ZnSO4 |
| 30 mM Sodium Acetate pH 4.6 |
|
3 ml 1 M NaOAc |
| 250 mM NaCl |
|
6.25 ml 4 M NaCl |
| |
|
0.35 ml H2O |
S1 Mix:
27.5 microliters 10X S1 Buffer
2.7 micrograms denatured salmon sperm DNA (a lab stock reagent)
85 units S1 nuclease (Gibco/BRL)
H2O to a final volume of 275 microliters/reaction
Stop Buffer:
6 microliters 0.25 M EDTA
2 micrograms tRNA (greater than 10 mg/ml)
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