Supplementary Protocol
DNase-chip protocol
Blunt end DNased DNA in-gel
(For low melt gel preparation of DNase treated DNA, see Crawford, et al., Genome Research 2006)
Wash extensively in T4 DNA Polymerase Buffer (+DTT)
3 x 50mls for 1 hour each (to get rid of EDTA in plug)
Remove all liquid from 50 ml conical tube and add Polymerase mix
DNA plug (low melt gel)
10x Polymerase Buffer (NEB#2)
dNTPs (10uM)
T4 DNA Polymerase
H20
BSA (100x)
80 ul
12
5
6 (NEB Cat # M0203L)
99.2
2
Incubate at room temp for 3-4 hours (gently mix every hour or so)
Add plug to 500 ul TE
Heat to 65 degrees for 10 minutes (flick every couple of minutes to dissolve agarose)
Phenol: Phenol/Chloroform: Chloroform extract (using wide-bore tips)
Add 1/10 volume 3M NaOAc, 2 volumes ETOH, and 1ul glycogen (Roche Cat# 901393)
Freezer for at least 30 minutes
Spin 15 minutes at 4 deg and wash 1x with 70% ETOH
Remove, quick spin, and remove remainder of liquid
Air dry for 5 minutes (NO LONGER!)
Resuspend in 40 ul TE
Ligation of biotinylated linkers
DNA (2ug)
H20
5x Ligase Buffer (use wide bore tips)
Annealed linkers (102B/103A)
T4 Ligase
Total
(2ug)
(to 50 ul total)
10 ul
(Invitrogen cat# 46300-018)
6.7 ul (THIS IS OLIGO SET A…THAW LINKERS ON ICE)
0.5 ul (NEB Cat # M0202L)
50 ul
Incubate O/N at 16 degrees
Shear DNA
Add ligation to 1.5 mls TE (in 15 ml conical)
Put on ice
Setting 3 for 25 second pulses (sonicate while tube is in ice bath)
Pulse each sample 8 times (back on ice after each sonication)
Tip should be almost at the bottom of tube, no movement
Cool sonicator tip after each round with ice bath
Bind to Streptavidin beads
Products
Dynal Streptavidin beads (Dynal M-280)
Dynal magnet (MPC-S cat# 120.20)
Use 100 ul of beads per reaction
FIRST Wash beads 3x 1ml in binding/wash buffer (TE + 1M NaCl)
1
Add 300 ul 5M NaCl to sonicated DNA (final concentration of 1M NaCl)
Add 100 ul of beads to sonicated DNA
Rock for 15 minutes at room temp
Use magnet to capture biotinylated ends
Wash 3 x 1ml in binding/wash buffer (TE + 1M NaCl)
Resuspend beads in blunt ending mix (see next section)
Blunt end sheared ends
H20
T4 Buffer (NEB buffer 2)
10 mM dNTPs
T4 DNA Polymerase
BSA (100x)
97.3 ul
11 ul
1 ul
(Roche 11 814 362 001)
0.2 ul (NEB Cat # M0203L)
0.5 ul
Incubate 1 hour at 16 degrees (resuspend beads once during incubation)
Wash beads 3 x 1ml in binding/wash buffer (TE + 1M NaCl)
Resuspend beads in 50 ul ligation mix
Ligation of non-biotinylated linkers to beads
Beads
H20
5x Ligase Buffer (use wide bore tips)
Annealed linkers (102C/103B)
T4 Ligase
Total
32.8 ul
10 ul
(Invitrogen cat# 46300-018)
6.7 ul (THIS IS OLIGO SET C...THAW LINKERS ON ICE)
0.5 ul (NEB Cat # M0202L)
50 ul
Add mix directly to beads and resuspend
Incubate 16 degrees O/N (resuspend beads once during incubation)
Clean Streptavidin beads
Wash 3 x 1ml in binding/wash buffer (TE + 1M NaCl)
Resuspend pellet in 50 ul TE pH 8.0
LM-PCR Reaction
H20
32.5
10x ThermoPol Buffer
4
dNTPs (10uM)
1.25
(Roche 11 814 362 001)
oligo OJW102 (40uM, 102C)
1.25
beads
1
(Heat up PCR machine to 50 deg, put on hold and add the following below)
Taq DNA Polymerase
H20
10x ThermoPol Buffer
Total
0.5
8.5
1
50 ul
(Invitrogen cat# 18038-042)
(NEB Cat #B90045)
Cycling Conditions
55 x 4 min (add Taq at this stage - hot start)
72 x 3 min
95 x 2 min
95 x 30 sec \
60 x 30 sec }>> 25 cycles
72 x 1 min /
2
72 x 5 min
4 x forever
Clean up of LM-PCR product
Add 400ul TE to pcr product
Phenol/chloroform extraction using phase locks 2.0 ml
(Brinkmann Instruments. Description: Eppendorf phase lock 2ml Cat # 62111-400)
ETOH ppt with 1ul glycogen (Roche Cat# 901393)
Resuspend pellet in 25 ul TE
Clean up on ArrayIT columns microarray probe purification kit (Cat #FPP $70 for 50 columns)
Elute with 50 ul water
Quantitate, label, and hybridize to arrays
Oligos
1) oJW102A: 5’ biotin TEG- GCG GTG ACC CGG GAG ATC TGA ATT C –3’
2) oJW102B: 5’ /5Bio/ GCG GTG ACC CGG GAG ATC TGA ATT C –3’
3) oJW102C: 5’ GCG GTG ACC CGG GAG ATC TGA ATT C –3’
4) oJW103A: 5’ /5Phos/GAA TTC AGA TC/3AmM/ -3’
5) oJW103B: 5’ GAA TTC AGA TC -3’
Oligo SET A: 102B/103A
Oligo SET C: 102C/103B
Protocol to anneal primers
50 ul Tris-HCL 1M, pH 7.6
375 ul of 40 uM oJW102
375 ul of 40 uM oJW103
200 ul H2O
TOTAL: 1000 ul
**Put mix in boiling water, shut off heat, and let oligos sit in water until room temp is reached, then put at 4 degrees
O/N. Aliquot and store at -20. To prevent primers from becoming un-annealed, always thaw annealed primers on
ice.
3