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In vitro translation Gel shift by YC (DOC)

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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                                   5.5ul
Oligo#1(100ng/ul)                     1ul
Gamma-ATP(32P)                        2ul of 1:10 diluted stock
10×Kinase buffer                      1.0ul
T4 PNK polynucleotide Kinase          0.5ul
Total                                 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               stock           vol(1ml)        vol(10ml)
200mM Hepes                     1M              200ul           2ml
30mM MgCl2                      1M              30ul            300ul
10mM DTT                        1M              10ul            100ul
10mM EDTA                       0.5M            20ul            200ul
H2O                                             740ul           7.4ml

2. Setting up rxns (a typical experiment will be something like below):

Reagents                retic con       DNA con         2A probe1       2D probe1
H2O                     20.5ul          23.5ul          20.5ul          20.5ul
dI/dc(1ug/ul):          1ul             1ul             1ul             1ul
10×binding buffer       3ul             3ul             3ul             3ul
50%Glycerol:            2.5ul           2.5ul           2.5ul           2.5ul
retic lysate:           3ul             -               -               -
mef2a retic lysate      -               -               3ul             -
mef2d retic lysate      -               -               -               3ul
Total                   30ul            30ul            30ul            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)                                5% Acrylamide gel
10× Buffer          1.2ul                                   40ml 1× TBE
50% Glycerol        1.2ul                                   10ml 40% 19:1 Acrylamide
ds dI/dC (1ug/ul)   1ul                                     30ml H2O
DTT (0.1M)          0.12ul [1mM final]                      350ul APS
MgCl2 (50mM)        0.36ul [1.5mM final]                    35ul TEMED

H2O            bring volume up to 11 ul after taking protein and extract volumes into account
Protein        approximately 1ul each
Extract        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                   5.5ul
Oligo#1(100ng/ul)     1ul
Gamma-ATP(32P)        2ul
10×Kinase buffer      1ul
Kinase                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):        1ul
retic lysate:         1.5ul
1×binding buffer      8.5ul
1×104 cpm probe       1ul
Total                 12ul

20min RT, 6% gel, load gel, 80v 45min

10×binding buffer             stock          volumn
200mM Hepes                   1M             200ul
30mM MgCl2                    1M             30ul
10mM DTT                      1M             10ul
10mM EDTA                     0.5M           20ul
H2O                                          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)                                5% Acrylamide gel
10× Buffer          1.2ul                                   40ml 1× TBE
50% Glycerol        1.2ul                                   10ml 40% 19:1 Acrylamide
ds dI/dC (1ug/ul)   1ul                                     30ml H2O
DTT (0.1M)          0.12ul [1mM final]                      350ul APS
MgCl2 (50mM)        0.36ul [1.5mM final]                    35ul TEMED

H2O            bring volume up to 11 ul after taking protein and extract volumes into account
Protein        approximately 1ul each
Extract        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.

				
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