LGT11 Screening for DNA-Binding Proteins
Library is in lgt11
Y1090r-: 20ml LB-Mg2+
0.2ml 20% maltose
10ml 100mg/ml Ampicillin
~100ml Y1090r- freeze
Grow overnight 37°C
Spin down Y1090r-, 10' ~3000 rpm. Resuspend pellets in 8ml 10mM MgSO4 (sterile). Keep on ice.
Prepare appropriate dilution(s) of phage in SM + gelatin.
1° ~ 50,000 pfu/plate
2° ~ 5,000 pfu/plate
3° ~ 100 pfu/plate
To 15 15ml tubes add 0.3ml Y1090r-.
Add phage in 10-30ml.
Incubate at 37°C 20'.
Add 8ml 53°C top agarose, pour onto 37°C LB-Mg2+ agar plates.
Cool at room temperature (rt) for 30'.
Incubate at 42°C for ~3 hrs or until small plaques are forming.
Overlay plate w/10mM IPTG-saturated nitrocellulose filters.
Incubate at 37° for ~4 hrs.
Mark filters w/ink and immerse in 200ml Blotto; rt, gentle shaking, 60'.
Wash 2 X 1' in TNE 50. (May need to vary salt between 10 and 100mM depending on particular probe.)
Hybridize 60' at rt with gentle shaking in 20 - 100ml with 2 X 106 dpm/ml of probe.
Wash 3 X 10' at rt with gentle shaking in TNE 50.
Expose filters to film overnight.
Probe: Oligos are kinased, annealed and ligated; then Nick translated.
SOLUTIONS
2L TNE 50 200ml Blotto
20ml 1M Tris pH7.5 10ml 1M Tris pH7.5
20ml 5M NaCl 2ml 5M NaCl
4ml 0.5M EDTA 0.4ml 0.5M EDTA
2ml 1M DTT 0.2ml 1M DTT
1954ml H2O 187.4ml H2O
10g Milk
Typical Hyb: 40ml TNE 50
200ml calf thymus DNA (1mg/ml)
400ml probe (2 X 106dpm/ml)
PREPARATION OF PROBE
2mg oligo 2mg oligo
5ml 10X Kinase / Ligation Buffer 5ml 10X Kinase / Ligation Buffer
2.5ml 100mM DTT 2.5ml 100mM DTT
5ml 10mM ATP 5ml 10mM ATP
2.5ml 10mM Spermidine 2.5ml 10mM Spermidine
H2O to 50ml H2O to 50ml
4ml T4 Polynucleotide Kinase (40U) 4ml T4 Polynucleotide Kinase (40U)
Incubate at 37°C 60'.
Mix reactions together.
Heat to 85o C, 2'.
Remove & place in 65°C H2O bath 15'.
Incubate at 37°C 15'.
Incubate at rt 15'.
Allow to cool on ice 15'.
Add 4ml (4U) T4 DNA ligase and 1ml 50mM ATP.
Incubate at 15°C overnight.
Stop reaction by adding 200ml 0.3M NaOAc, 5mM EDTA, 0.5% SDS.
Phenol: chloroform extract.
Chloroform: Isoamyl Alcohol extract.
Precipitate with 3 vol. 95% Ethanol.
Wash with 500ml 70% Ethanol.
Resuspend in 24ml TE (167ng/ml).
Run some on 3% Nu Sieve gel to confirm ligation (should see a ladder or a smear).
Nick Translate 250ng using BRL kit.
Must purify over a 5ml Sephadex G50 drip column, not a spin column.
10X Kinase / Ligation Buffer
0.5M Tris pH7.5
0.1M MgCl2
» Top