A | B | C | D | E | F | |
Reagent | 1X | 10% extra | 3X | 5X | 8X | |
UltraPure water | 40 ul | 44 ul | 132 ul | 220 ul | 352 ul | |
10x Nuclease digest buffer | 5 ul | 5.5 ul | 16.5 ul | 27.5 ul | 44 ul | |
100 mM MgCl2 | 5 ul | 5.5 ul | 16.5 ul | 27.5 ul | 44 ul | |
total | 50 ul | 165 ul | 275 ul | 440 ul |
A | B | |
Temp | Time | |
55 | 15 minutes | |
68 | 45 minutes | |
25 | Hold |
A | B | C | D | E | F | |
Reagent | 1X | 10% extra | 3X | 5X | 8X | |
UltraPure water | 45 ul | 49 ul | 147 ul | 245 ul | 392 ul | |
10x Crosslink Reversal buffer | 5 ul | 5.5 ul | 16.5 ul | 27.5 ul | 44 ul | |
Proteinase K | 1.5 ul | 1.65 ul | 4.95 ul | 8.25 ul | 13.2 ul | |
total | 51.5 ul | 165 ul | 275 ul | 440 ul |
A | B | |
Temp | Time | |
55 | 15 minutes | |
68 | 45 minutes | |
25 | hold |
A | B | C | D | E | F | |
Reagents | 1X | 10% extra | 3X | 5X | 8X | |
End Polish Buffer | 50 ul | 55 ul | 165 ul | 275 ul | 440 ul | |
End Polishing Enzyme Mix | 3.5 ul | 3.85 ul | 11.55 ul | 19.25 ul | 30.8 ul | |
Total | 53.5 ul |
A | B | |
Temp | Time | |
22 | 30 minutes | |
65 | 30 minutes |
A | B | C | D | E | F | |
Reagents | 1X | 10% extra | 3X | 5X | 8X | |
UltraPure water | 35 ul | 38.5 ul | 115.5 ul | 192.5 ul | 308 ul | |
5X bridge ligation buffer | 10 ul | 11 ul | 33 ul | 55 ul | 88 ul | |
Bridge | 5 ul | 5.5 ul | 16.5 ul | 27.5 ul | 44 ul | |
Total | 53.5 ul |
A | B | |
Reagent | Volume/reaction | |
Bridge ligation mix | 50 ul | |
Bridge ligase | 1 ul | |
Total | 51 ul |
A | B | C | D | E | F | |
Reagents | 1X | 10% extra | 3X | 5X | 8X | |
Intra-Aggregate Ligation Buffer | 50 ul | 55 ul | 165 ul | 275 ul | 440 ul | |
Intra-Aggregate Ligation enzyme mix | 2 ul | 2.2 ul | 6.6 ul | 11 ul | 17.6 ul | |
Total | 53.5 ul |
A | B | C | D | E | F | |
Reagents | 1X | 10% extra | 3X | 5X | 8X | |
UltraPure water | 45 ul | 49.5 ul | 148.5 ul | 247.5 ul | 396 ul | |
10% crosslink reversal buffer | 5 ul | 5.5 ul | 165 ul | 275 ul | 44 ul | |
Proteinase K | 1.5 ul | 1.65 ul | 4.95 ul | 8.25 ul | 13.2 ul | |
Total | 51.5 ul |
A | B | |
Temp | Time | |
55 | 15 minutes | |
68 | 45 minutes | |
25 | hold |
A | B | C | D | E | F | |
Reagents | 1X | 10% extra | 3X | 5X | 8X | |
End Repair Buffer | 7 ul | 7.7 ul | 23.1 ul | 38.5 ul | 61.6 ul | |
End repair enzyme mix | 3 ul | 3.3 ul | 9.9 ul | 16.5 ul | 26.4 ul | |
250 mM DTT | 0.5 ul | 0.55 ul | 1.65 ul | 2.75 ul | 4.4 ul | |
Total | 10.5 ul |
A | B | |
Temp | Time | |
20 | 30 minutes | |
65 | 30 minutes | |
12 | hold |
A | B | |
Reagent | Volume/reaction | |
Adaptor for Illumina | 2.5 ul | |
Ligation Enzyme mix | 30 ul | |
Ligation Enhancer | 1 ul | |
Total | 33.5 ul |
A | B | C | D | E | F | |
Reagents | 1X | 10% extra | 3X | 5X | 8X | |
UltraPure water | 15 ul | 16.5 ul | 49.5 ul | 82.5 ul | 132 ul | |
HotStart PCR Ready Mix | 25 ul | 27.5 ul | 82.5 ul | 137.5 ul | 220 ul | |
Universal PCR primer | 5 ul | 5.5 ul | 16.5 ul | 27.5 ul | 44 ul | |
Total | 45 ul |
A | B | C | D | |
Step | Temp | Time | Cycles | |
Enzyme activation | 98 | 3 minutes | 1 | |
Denature | 98 | 20 seconds | 12 | |
Anneal | 65 | 30 seconds | ||
Extend | 72 | 30 seconds | ||
Extend | 72 | 1 minute | 1 | |
12 | hold | 1 |