1. Wipe test: Using small
circular paper, wipe inside box (1), plastic tray (2), outside yellow vial (3),
and inside yellow vial (4). Put each paper into scintialliation vial labeled
1-4 and count with scintillation counter in Shearn lab. Use program 9, user 1.
Counts should be <25 cpm. Record (1), (2), and (4) on radiation form.
2. Label oligos:
(1) 8 bp 5Õ + nic + 40 bp 3Õ (.033 nmol/ml) 10mM: 1.5 ml
.033 nmol/ml + 3.5 ml dH20
(2) 24 bp 5Õ + nic + 24 bp
3Õ (.036 nmol/ml) 10mM: 1.4 ml
.036 nmol/ml + 3.6 ml dH20
(3) 40 bp 5Õ + nic + 8 bp 3Õ
(.026 nmol/ml) 10mM: 1.9ml
.026 nmol/ml + 3.1 ml dH20
(c1) R&C 24 bp 5Õ + nic
+ 24 bp 3Õ (.017 nmol/ml) 10mM: 2.9 ml .017 nmol/ml +
2.1 ml dH20
(c2) PamÕs TraY OriT (.017 nmol/ml) 10mM: 2.9 ml
.017 nmol/ml + 2.1 ml dH20
Prepare the following
reaction:
Incubate @ 37¡C 45 min
Incubate @ 70¡C 10 min
Cool briefly, spin briefly
+ 30 ml sterile dH20
+ 50 ml phenol: chloroform: isoamyl alcohol
Vortex, spin 2 min
Prepare Spin column
Invert column several times
to resuspend
Sit column over collection
tube
Spin @ 2200 rpm 1 min
Discard flowthru and place
column in new tube
Apply aqueous layer over
spin column and spin 2 min @ 2200 rpm
1:50 dilution
Read counts of 2 ml of 1:50 dilution
3. Prepare the following
buffer to a total volume of 50 ml:
4. To 1ml of the above
buffer, add 1 mg/ml calf DNA.
5. Prepare the following
solutions to a total volume of 50ml:
DNA
in H20
DNA
in 0 nM TraI36
DNA
in 100 nm TraI36
TraI36 dilution: 88.96mM initial concentration
100nM: 3.4ml (8.896mM TraI36)+ 296.6ml (buffer + calf DNA) = 300 ml
0 nM: 300ml buffer + calf DNA = 300 ml
6. Incubate 50, 000 cpm
oligos with TraI36 for 1.5 hours @ RT. (3:30-5 pm)
|
|
DNA (ml) 1:50 dilution |
Protein |
||
|
|
|
TraI36 (88.96mM) |
Buffer + calf DNA |
H2O |
|
Oligo 1 in H20 |
3.5 |
0 |
0 |
46.5 |
|
Oligo 1 in 0 nM TraI36 |
3.5 |
0 |
46.5 |
0 |
|
Oligo 1 in 1000 nM TraI36 |
3.5 |
46.5 |
0 |
0 |
|
|
|
|
|
|
|
Oligo 2 in H20 |
8.2 |
0 |
0 |
41.8 |
|
Oligo 2 in 0 nM TraI36 |
8.2 |
0 |
41.8 |
0 |
|
Oligo 2 in 1000 nM TraI36 |
8.2 |
41.8 |
0 |
0 |
|
|
|
|
|
|
|
Oligo 3 in H20 |
1.8 |
0 |
0 |
48.2 |
|
Oligo 3 in 0 nM TraI36 |
1.8 |
0 |
48.2 |
0 |
|
Oligo 3 in 1000 nM TraI36 |
1.8 |
48.2 |
0 |
0 |
|
|
|
|
|
|
|
Oligo C(1) in H20 |
7.5 |
0 |
0 |
42.5 |
|
Oligo C(1) in 0nM TraI36 |
7.5 |
0 |
42.5 |
0 |
|
Oligo C(1) in 1000 nM
TraI36 |
7.5 |
42.5 |
0 |
0 |
|
|
|
|
|
|
|
Oligo C(2) in H20 |
16 |
0 |
0 |
34 |
|
Oligo C(2) in 0nM TraI36 |
16 |
0 |
34 |
0 |
|
Oligo C(2) in 1000 nM
TraI36 |
16 |
34 |
0 |
0 |
7. Add to cap of tube:
2 ml (125 mM Fe (II) + 250 mM EDTA)
2 ml 28 mM NaAscorbate
2 ml 0.8% H202
Close tube and spin down
solutions.
8. Incubate 2 min @ RT.
9. Stop rxn by adding 150 ml stop solution to each tube.
1 sample 15
+ 1 samples
112 ml TE 1792
ml TE
10 ml 0.1M thiourea 160
ml 0.1M thiourea
25 ml 3M NaAc 400
ml 3M NaAc
2 ml 0.2M EDTA 32
ml 0.2M EDTA
5 ml 1 mg/ml tRNA 80
ml 1 mg/ml tRNA
9. Extract w/ phenol
chloroform
Add equal volume of bottom
layer of phenol: chloroform
Vortex
Spin 2 min @ max speed
Take off top layer
Repeat 1x
10. Ethanol precipitate
+ 10% 3M NaAc (.1 x 200=20ml)
+ 2.5x 95% EtOH (2.5 x
200=500ml)
Sit @ -80¡C > 15 min
Spin max speed @ 4¡C 15 min
Wash w/ 20 ml 70% EtOH
Sit @ -20¡C 5 min
Spin max @ 4¡C 8 min
Aspirate 70% EtOH
Speed vac 15 min
Dissolve in 5ml TE + 5ml formamide loading buffer.
11. Read counts.
12. Prepare 12% sequencing
gel with thin spacers and 20-well comb.
50 ml 12% sequencing gel
24 ml SequaGel concentrate
21 ml SequaGel diluent
5 ml SequaGel buffer
400 ml 10% APS
20 ml TEMED
Rinse wells with 1xTBE.
Pre-run gel at 300 V 30 min.
Load 3ml sample/lane. Load G-reaction of each labeled oligo.
Run at 300-500 V 2h.
13. Soak gel with bottom
glass plate in 10% MeOH + 10% acetic acid 45 min.
14. Dry gel 1h 15 min.
Expose overnight in phosphoimager casette.
15. Develop 16 hours later in Schleif lab.