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Revised 200.6.1
Long Range Footprinting Protocol (SP1)

Based on Bio-Techniques,23, 300-303 (1997)

a) Reagent required

    pSI Mammalian Expression Vector (Promega)

    Dynabeads M-280 Streptavidin (DYNAL)

    MPC-M (DYNAL)

    TaKaRa Ex Taq (Takara)

    MicroSpin S-400 HR Columns (Pharmacia Biotech)

    IRD-41 Labeled Custom Primer (Aloka)

    Biotinylated Primer

    SP1:human (Promega)

    DNase I FPLC Pure (Pharmacia Biotech)

     

    b) Preparation of reagents

    5 x RM buffer

25 mM HEPES (pH 7.8) 0.25M HEPES 1 ml
50 mM KCl 5M KCl 100 ml
0.05 mM EDTA 250mM EDTA 2 ml
0.5 mM DTT 1M DTT 5 ml
0.5 mM PMSF 1M PMSF 5 ml
5% glycerol glycerol 500 ml
　 dH2O 8.39 ml
　 Total 10 ml
　

　

Stop Solution

4 M NaCl 5 M NaCl 32 ml
100 mM EDTA 0.5 M EDTA 8 ml
　 Total 40 ml
　

　

BW buffer (Binding & Washing buffer)

10 mM Tris-HCl (pH 7.5) 1 M Tris-HCl 500 ml
1 mM EDTA 0.5 M EDTA 100 ml
2 M NaCl 5 M NaCl 20 ml
　 dH2O 29.4 ml
　 Total 50 ml

　

c) Preparation of probe DNA immobilized on the beads

1) PCR 
pSI Mammalian Expression Vector (SV40EP DNA) 50 ng
dNTP mix 5 ml
10 x ExTaq buffer 5 ml
Biotinylated Primer (20mM) 0.5 ml
IRD41 labeled Primer 10 ml
ExTaq 0.5 ml
dH2O x ml
Total 50 ml

PCR Program: 16 Cycles

94℃ 1 min
37℃ 1 min
72℃ 1 min

　

2) Purify the amplified DNA through a S-400 HR column 

3) Take 60ml of Dynabeads M-280 streptavidin beads in a 1.5 ml Eppendorf tube, stand on Dynal MPC-M for 30 sec and remove supernatant.

4) Add 100ml of BW buffer, wash the beads and remove supernatant. Repeat once.

5) Suspend the beads in 50ml of BW buffer and add 50ml of the S-400HR-purified DNA.

6) Incubate the reaction at room temperature for 60 min with occasional mixing.

7) Remove supernatant, wash the beads with 100ml of BW buffer and remove supernatant. Repeat once.

8) Suspend the beads with 30ml of 1 x RM buff

　

 d) Footprinting reaction

1) Mix the following reagents

 
DNA immobilized on the beads 4ml
5 x RM buffer (with 5 mM MgCl2) 20ml
SP1 (1 fpu/ml) 1.5ml
dH2O 74.5ml
Total 100ml

 

2) Incubate the reaction at room temperature for 30 min with occasional mixing.

3) Add 0.75 units of DNase I, incubate for 60 sec, immediately add 100 ml of Stop Solution and place the tube on ice.

4) Place the tube on Dynal MPC-M and remove supernatant.

5) Add 100 ml of BW buffer and remove supernatant.

6) Add 100 ml of TE and remove supernatant.

7) Add 1.5 ml of Loading Dye Solution (contained in a sequencing kit), denature at 95oC for 2 min and immediately chill on ice.

e) Electrophoresis

Urea 25.2 g
Long Ranger 6.6 ml
water 26 ml
　
Electrophoresis conditions

　
Running buffer 0.5 x TBE
Voltage 2250 V
Current 30.6 mA
Power 68.8 W
Heater 50oC
Motor speed 4
Signal channel 3
Frame 25

　

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