Labeling DNA anchoring fragments
Reagent	Concentration	Volume (µL)
pBluescriptKS	250 ng/µL	1
Primer A (CTAAATTGTAAGCGTTAATATTTTGTTAAA)	100 µM	1
Primer B, (TATCTTTATAGTCCTGTCGGGTTTCGCCAC)	100 µM	1
dNTPs	10 mM	1.5
Mg2+	25 mM	2
Taq buffer without Mg2+	10×	5
Taq polymerase	Manufacturer stock (NEB)	1
Digoxigenin-11-dUTP or biotin-16-dUTP (Roche)	1 mM	1.5
DI H2O		36.5

PCR program for DNA labeling
Step	T (°C)	Duration (min)	Number of cycles
1	94	5	1
	94	0.5	30
2	54	1
	72	1
3	72	5	1

Central fragment digestion
Reagent	Volume (µL)
pFX355 (10 kbp at 100 ng/µL)	10
XhoI	1
AatII	1
Eco109I	1
NEB 4	3
H2O	14
37 °C for 1 h + 65 °C for 20 min

DIG-labeled fragment digestion
Reagent	Volume (µL)
DIG fragment (~1 kbp at 50 ng/µL)	10
XhoI	2
NEB buffer 4	4
H2O	4
37 °C for 1 h + 65 °C for 20 min

Biotin-labeled fragment digestion
Reagent	Volume (µL)
Biotin fragment (~1 kbp at 50 ng/µL)	10
AatII	2
NEB buffer 4	4
H2O	4
37 °C for 1 h + 65 °C for 20 min

Ligation of anchoring fragments to DNA central sequence
Reagent	Volume (µL)
Digested central fragment	2
Digested DIG fragment	15
Digested Biotin fragment	15
Ligase buffer 10×	10
T4 DNA ligase (Fermentas)	4
H2O	52
16 °C for 2 h then 65 °C for 20 min