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In vitro translation and Gel Shift
In vitro translation (from Laurie)
Note: before starting, dilute 35S met 1ul+4ul, use 1ul in 5ul rxn.
H2O:
9ul
Retic lystate: 12.5ul
Buffer:
1ul
SP6 RNA pol: 0.5ul
AA- met:
0.5ul
RNasin:
0.5ul
Template:
1ul
Total:
25ul
*Take 5ul of the above rxn, add 1ul of diluted 35S
*for the rest 20ul rxn, add 0.5ul AA-leu (this is to compensate the methionine in AA-met)
Incubate 30 degree for 2h
Freeze the non-labeled rxn.
Check 2ul of the 35S reactions on SDS-PAGE
Dry gel: put ethanol and dry ice in the bucket, and set the timers for heat and vacuum for 1h.
Expose to film.
Labelling Oligo probe for gel shift (from Laurie)
Note: before starting, dilute the 166 uCi/ul gamma ATP stock 1:10
H2O
Oligo#1(100ng/ul)
Gamma-ATP(32P)
10×Kinase buffer
T4 PNK polynucleotide Kinase
Total
5.5ul
1ul
2ul of 1:10 diluted stock
1.0ul
0.5ul
10ul
Incubate 37°C, 30min → Heat 95°C, 2min
→ Add 1.1ul 0.5M NaCl (final concentration of NaCl is 50mM)
→ Add oligo #2 (3ul) (3× as much as #1, 300ng) → Re-heat to 95°C, 2min
Cooling slowly to anneal oligos by placing tube (stick through foil) into beaker of boiling water
and allow cool to RT on bench
Add H2O to 50ul
Purifing the probes with G-50 / G-25 columns:
Note: For G-25, labeled DNA should be at least 10 bases in length; For G-50, labeled DNA
should be at least 20 bases in length.
1) Resuspend the resin by vortexing
2) Loose the cap ¼, snap off the closure, place the column on a microcentrifuge tube, and
centrifuge for 1min at 3000rpm(to avoid damaging the beads, this speed is desired)
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3) Discard the cap of the column, place the column in a new microcentrifuge tube
4) Apply the sample to the column, and centrifuge for 2min at 3000rpm(important)
If the probe is hot enough, dilute it 1:10 in water, and use 1ul/rxn. If it is not hot, discard it and
remake new ones.
Setting up Gel Shift Reactions:
1. before you start:
1) Prepare 6% Acrylamide gel (total 80ml)
40ml 1× TBE
12ml 40% 19:1 Acrylamide (important for good resolution)
28ml H2O
350ul APS
35ul TEMED
2) Make sure there is enough 10X binding buffer
10×binding buffer
200mM Hepes
30mM MgCl2
10mM DTT
10mM EDTA
H2O
stock
1M
1M
1M
0.5M
vol(1ml)
200ul
30ul
10ul
20ul
740ul
vol(10ml)
2ml
300ul
100ul
200ul
7.4ml
2. Setting up rxns (a typical experiment will be something like below):
Reagents
H2O
dI/dc(1ug/ul):
10×binding buffer
50%Glycerol:
retic lysate:
mef2a retic lysate
mef2d retic lysate
Total
retic con
20.5ul
1ul
3ul
2.5ul
3ul
30ul
DNA con
23.5ul
1ul
3ul
2.5ul
30ul
2A probe1
20.5ul
1ul
3ul
2.5ul
3ul
30ul
2D probe1
20.5ul
1ul
3ul
2.5ul
3ul
30ul
Combine all the components above, and incubate at 30°C for 15mins
Add the 1:10 diluted probe as following
1×104 cpm probe
1ul
1ul
1ul
1ul
Incubate at RT for 10mins
Load gel : lane #1 should be dye alone
Run the gel IN THE COLD ROOM, 200v, 2-3h, until the dye goes 2/3 of the gel
Dry the gel and expose to film O/N
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Below are the protocols from Karen.
Labeling probe(from Karen)
1 ul
oligo#1 (25pmol/ul)
5.5 ul
H2O
0.5ul
gamma ATP
1ul
kinase buffer
1ul
kinase
Incubate at 37C for 30-60 min. Bring volume up to 30-40ul with H2O. Spin through G-50
column. Heat kill enzyme for 3min at 95C. Spin. Add 2ul Oligo#2 (25pmol/ul). Heat to 95C for
3 min. Cool slowly to RT.
Use 0.1ul probe or approximately 50,000cpm in each reaction.
Buffer (10×)
200mM Hepes, PH 7.6
500mM KCl
10mM EDTA
Reaction Mix (12ul reaction)
10× Buffer
1.2ul
50% Glycerol
1.2ul
ds dI/dC (1ug/ul)
1ul
DTT (0.1M)
0.12ul [1mM final]
MgCl2 (50mM)
0.36ul [1.5mM final]
H2O
Protein
Extract
5% Acrylamide gel
40ml 1× TBE
10ml 40% 19:1 Acrylamide
30ml H2O
350ul APS
35ul TEMED
bring volume up to 11 ul after taking protein and extract volumes into account
approximately 1ul each
approximately 1ul each
Make up master mix with above reagents. If required, add proteins and extract individually to
each reaction. Mix by finger tapping.
Incubate proteins, extract, and other reagents at 37C for 20min.
Add probe (0.1ul plus 0.9ul H2O)
Incubate at RT for 15min
Place on ice before loading
Load entire reaction on 5% Acrylamide gel made with 0.5× TBE buffer.
Run at 160 volts for 2-3 hours in 0.5× TBE buffer (Probe runs off at 2.5 hours).
Expose overnight at -80C.
Labelling Oligo probe for gel shift (from Karen)
Combine
H2O
Oligo#1(100ng/ul)
Gamma-ATP(32P)
10×Kinase buffer
Kinase
5.5ul
1ul
2ul
1ul
0.5ul
10ul
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Incubate 37°C, 30min
Heat 95°C, 2min
Add NaCl to 50mM (0.6ul of 1M NaCl)
Add oligo #2 (3ul) (3× as much as #1, 300ng)
Re-heat to 95°C, 2min 3ul
Cooling slowly to anneal oligos by placing tube (stick through foil) into beaker of 65°C water
and allow cool to RT on bench
Add H2O to 50ul
G-50 column
Count 1ul=1.0×105 cpm/ul
(Charlotte: 50,000 counts/reaction; Laurie: Dilute 1/10, use 1ul/rxn)
CAB1/CAB2 do not contain end GAT seq used for polymerization of oligos
Reaction:
dI/dc(1ug/ul):
retic lysate:
1×binding buffer
1×104 cpm probe
Total
1ul
1.5ul
8.5ul
1ul
12ul
20min RT, 6% gel, load gel, 80v 45min
10×binding buffer
200mM Hepes
30mM MgCl2
10mM DTT
10mM EDTA
H2O
stock
1M
1M
1M
0.5M
volumn
200ul
30ul
10ul
20ul
740ul
Labeling probe
1 ul
oligo#1 (25pmol/ul)
5.5 ul
H2O
0.5ul
gamma ATP
1ul
kinase buffer
1ul
kinase
Incubate at 37C for 30-60 min. Bring volume up to 30-40ul with H2O. Spin through G-50
column. Heat kill enzyme for 3min at 95C. Spin. Add 2ul Oligo#2 (25pmol/ul). Heat to 95C for
3 min. Cool slowly to RT.
Use 0.1ul probe or approximately 50,000cpm in each reaction.
Buffer (10×)
200mM Hepes, PH 7.6
500mM KCl
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10mM EDTA
Reaction Mix (12ul reaction)
10× Buffer
1.2ul
50% Glycerol
1.2ul
ds dI/dC (1ug/ul)
1ul
DTT (0.1M)
0.12ul [1mM final]
MgCl2 (50mM)
0.36ul [1.5mM final]
H2O
Protein
Extract
5% Acrylamide gel
40ml 1× TBE
10ml 40% 19:1 Acrylamide
30ml H2O
350ul APS
35ul TEMED
bring volume up to 11 ul after taking protein and extract volumes into account
approximately 1ul each
approximately 1ul each
Make up master mix with above reagents. If required, add proteins and extract individually to
each reaction. Mix by finger tapping.
Incubate proteins, extract, and other reagents at 37C for 20min.
Add probe (0.1ul plus 0.9ul H2O)
Incubate at RT for 15min
Place on ice before loading
Load entire reaction on 5% Acrylamide gel made with 0.5× TBE buffer.
Run at 160 volts for 2-3 hours in 0.5× TBE buffer (Probe runs off at 2.5 hours).
Expose overnight at -80C.
Walsh TBE (20X)
For 2L
1M Tris
242.2g
1M Boric Acid
123.7g
20mM EDTA(Na4)
15.2g
sGDW
to volume
Preparation of Poly(dI-dC).Poly(dI-dC) — Amersham Pharmacia #27-7880
-resuspend in 1× gel shift buffer (20mM HEPES, 50mM KCl, 1mM EDTA, 1.5mM MgCl2) to a
concentration of 0.5-2ug/ul.
-Heat to 45C for 5 min, anneal slowly.
-store at -20C in small aliquots.