Labelling Oligo Probe for Gel Shift

In vitro translation & Gel shift.doc

Page 1 of 5

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

H2O5.5ul

Oligo#1(100ng/ul)1ul

Gamma-ATP(32P)2ul of 1:10 diluted stock

10×Kinase buffer1.0ul

T4 PNK polynucleotide Kinase0.5ul

Total10ul

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)

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:

before you start:

1)Prepare 6% Acrylamide gel (total 80ml)

40ml 1× TBE

12ml 40% 19:1Acrylamide (important for good resolution)

28ml H2O

350ul APS

35ul TEMED

2) Make sure there is enough 10X binding buffer

10×binding bufferstockvol(1ml)vol(10ml)

200mM Hepes1M 200ul2ml

30mM MgCl21M30ul300ul

10mM DTT1M10ul100ul

10mM EDTA0.5M20ul200ul

H2O740ul7.4ml

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

Reagentsretic con DNA con2A probe12D probe1

H2O20.5ul23.5ul20.5ul20.5ul

dI/dc(1ug/ul):1ul1ul1ul1ul

10×binding buffer3ul3ul3ul3ul

50%Glycerol:2.5ul2.5ul2.5ul2.5ul

retic lysate:3ul---

mef2a retic lysate--3ul-

mef2d retic lysate---3ul

Total30ul30ul30ul30ul

Combine all the components above, and incubate at 30°C for 15mins

Add the 1:10 diluted probe as following

1×104 cpm probe1ul1ul1ul1ul

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

Below are the protocols from Karen.

Labeling probe(from Karen)

1 uloligo#1 (25pmol/ul)

5.5 ulH2O

0.5ulgamma ATP

1ulkinase 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× Buffer1.2ul40ml 1× TBE

50% Glycerol1.2ul10ml 40% 19:1 Acrylamide

ds dI/dC (1ug/ul)1ul30ml H2O

DTT (0.1M)0.12ul [1mM final]350ul APS

MgCl2 (50mM)0.36ul [1.5mM final]35ul TEMED

H2Obring volume up to 11 ul after taking protein and extract volumes into account

Proteinapproximately 1ul each

Extractapproximately 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

H2O5.5ul

Oligo#1(100ng/ul)1ul

Gamma-ATP(32P)2ul

10×Kinase buffer1ul

Kinase0.5ul

10ul

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 buffer8.5ul

1×104 cpm probe1ul

Total12ul

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

10×binding bufferstockvolumn

200mM Hepes1M 200ul

30mM MgCl21M30ul

10mM DTT1M10ul

10mM EDTA0.5M20ul

H2O740ul

Labeling probe