2.3.2 qPCR
1. Primers at 5 μM: For 100 μL, mix 5 μL of forward primer at
100 μM and 5 μL reverse primer at 100 μM to 90 μL ultrafiltrated H 2 O.
2. Primers in SYBR green mix: per well, mix 3 μL iTaq Universal
SYBR Green supermix with 0.25 μL of the primer mix at 5 μM
(see Note 14).
3. cDNA: dilute cDNA product in RNase-free water to reach
40 ng in a volume of 2.8 μL. Prepare for N + 5 wells and
keep on ice.
2.4 Material
for DNA-FISH
2.4.1 DNA-FISH Probes
Preparation
Our DNA-FISH probes are composed of fluorescently labeled
small DNA fragments (from 150 to 1000 bp) custom-prepared
using BACs and fosmids identified via the UCSC genome browser.
1. BAC/fosmid DNA at a concentration of 100–200 ng/μL.
Stored in H 2 O at À20
C (see Note 15).
2. 1 M Tris–HCl pH 7.5: for 100 mL, in a beaker, dissolve 12.1 g
Tris base in approximately 80 mL H 2 O. Add 37% HCl very
slowly up to pH ¼ 7.5 (see Note 4). Complete with H 2 O up to
100 mL with a graduated cylinder.
3. 1 M MgCl 2 : for 100 mL, in a beaker, dissolve 20.3 g
MgCl 2
. 6H 2 O into H 2 O and complete with H 2 O up to
100 mL with a graduated cylinder.
4. 10 mg/mL Bovine Serum Albumine (BSA): for 10 mL, dissolve 100 mg BSA in 10 mL H 2 O. Store as 1 mL aliquots at
À20
C.
5. 10Â Nick Translation Buffer (NTB): 0.5 M Tris–HCl
(pH 7.5), 0.05 M MgCl 2 , 0.5 mg/mL BSA. For 10 mL,
combine 5 mL 1 M Tris–HCl pH 7.5, 500 μL 1 M MgCl 2
and 500 μL BSA at 10 mg/mL with 4 mL H 2 O. Store as 1 mL
aliquots at À20
C.
6. 1 M DTT stock solution: dissolve 1 g DTT into 6.48 mL of
H 2 O and store at À20
C. Prepare 0.1 M DTT by diluting
tenfold 1 M DTT into H 2 O. Store as 50 μL aliquots at À20
C.
7. 10Â dNTP mix: 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM
dGTP, 0.125 mM dTTP. For 1 mL, combine 5 μL dATP at
100 mM, 5 μL dCTP at 100 mM, 5 μL dGTP at 100 mM,
1.25 μL dTTP at 100 mM. Store as 50 μL aliquots at À20
C.
8. DNase I (0.2 U/μL).
9. DNA Polymerase I (10 U/μL).
10. 0.5 mM 5-(3-Aminoallyl)-dUTP: for 500 μL, combine 5 μL of
5-(3-Aminoallyl)-dUTP with 495 μL in H 2 O. Store as 20 μL
aliquots at À80
C.
11. 1Â TAE: dilute 200 mL of 50Â TAE in 10 L H 2 O.
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1. Primers at 5 μM: For 100 μL, mix 5 μL of forward primer at
100 μM and 5 μL reverse primer at 100 μM to 90 μL ultrafiltrated H 2 O.
2. Primers in SYBR green mix: per well, mix 3 μL iTaq Universal
SYBR Green supermix with 0.25 μL of the primer mix at 5 μM
(see Note 14).
3. cDNA: dilute cDNA product in RNase-free water to reach
40 ng in a volume of 2.8 μL. Prepare for N + 5 wells and
keep on ice.
2.4 Material
for DNA-FISH
2.4.1 DNA-FISH Probes
Preparation
Our DNA-FISH probes are composed of fluorescently labeled
small DNA fragments (from 150 to 1000 bp) custom-prepared
using BACs and fosmids identified via the UCSC genome browser.
1. BAC/fosmid DNA at a concentration of 100–200 ng/μL.
Stored in H 2 O at À20
C (see Note 15).
2. 1 M Tris–HCl pH 7.5: for 100 mL, in a beaker, dissolve 12.1 g
Tris base in approximately 80 mL H 2 O. Add 37% HCl very
slowly up to pH ¼ 7.5 (see Note 4). Complete with H 2 O up to
100 mL with a graduated cylinder.
3. 1 M MgCl 2 : for 100 mL, in a beaker, dissolve 20.3 g
MgCl 2
. 6H 2 O into H 2 O and complete with H 2 O up to
100 mL with a graduated cylinder.
4. 10 mg/mL Bovine Serum Albumine (BSA): for 10 mL, dissolve 100 mg BSA in 10 mL H 2 O. Store as 1 mL aliquots at
À20
C.
5. 10Â Nick Translation Buffer (NTB): 0.5 M Tris–HCl
(pH 7.5), 0.05 M MgCl 2 , 0.5 mg/mL BSA. For 10 mL,
combine 5 mL 1 M Tris–HCl pH 7.5, 500 μL 1 M MgCl 2
and 500 μL BSA at 10 mg/mL with 4 mL H 2 O. Store as 1 mL
aliquots at À20
C.
6. 1 M DTT stock solution: dissolve 1 g DTT into 6.48 mL of
H 2 O and store at À20
C. Prepare 0.1 M DTT by diluting
tenfold 1 M DTT into H 2 O. Store as 50 μL aliquots at À20
C.
7. 10Â dNTP mix: 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM
dGTP, 0.125 mM dTTP. For 1 mL, combine 5 μL dATP at
100 mM, 5 μL dCTP at 100 mM, 5 μL dGTP at 100 mM,
1.25 μL dTTP at 100 mM. Store as 50 μL aliquots at À20
C.
8. DNase I (0.2 U/μL).
9. DNA Polymerase I (10 U/μL).
10. 0.5 mM 5-(3-Aminoallyl)-dUTP: for 500 μL, combine 5 μL of
5-(3-Aminoallyl)-dUTP with 495 μL in H 2 O. Store as 20 μL
aliquots at À80
C.
11. 1Â TAE: dilute 200 mL of 50Â TAE in 10 L H 2 O.
358
Noe ´ mie Kempf et al.