HEPES, pH 7.2 (1M) | FisherScientific | AAJ16924K2 | |
NaCl (5M) | Invitrogen | AM9760G | |
EDTA (0.5M) | Invitrogen | AM9260G | |
Triton X-100 (10%) | VWR | 97063-864 | |
DTT (1M) | Krackeler | 45-43816-50ML | |
Glycerol (100%) | VWR | MK509202 | |
Nuclease-free water | Invitrogen | AM9932 | |
Tn5 or TDE1 | homemade or Illumina | 20034197 | |
SDS (10%) | Invitrogen | 15553027 | |
pUC19 | NEB | N3041S | |
EcoRI | NEB | R3101S | |
Zymo Research Clean and Concentrate-5 | Zymo | D4014 | |
LUNA 2x qPCR master mix | NEB | M3003S | |
Qubit 1X dsDNA HS Assay Kit | Invitrogen | Q33231 |
A | B | |
Tn5ME_Rev | /5Phos/CTGTCTCTTATACACATCT | |
Tn5ME-A (Illumina FC-121-1030) | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG | |
Tn5ME-B (Illumina FC-121-1031) | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG |
A | B | C | |
Name | Sequence | pUC19 location(bp) | |
591bp_pUC19_Tn5_F | GCTCACTCAAAGGCGGTAAT | 748-1319 | |
591bp_pUC19_Tn5_R | CTTCAGCAGAGCGCAGATAC | ||
602bp_pUC19_Tn5_F | CTTTCACCAGCGTTTCTGGG | 2396-248 | |
602bp_pUC19_Tn5_R | GCTGGCGTAATAGCGAAGAG | ||
610bp_pUC19_Tn5_F | CCTATCTCAGCGATCTGTCTATTTC | 1651-2241 | |
610bp_pUC19_Tn5_R | GCGCGGTATTATCCCGTATT |
A | B | C | D | E | F | G | |
Oligo Concentration(uM) | 1.25 | 2.5 | 5 | 10 | 20 | 40 | |
Oligo(uL) | 1 | 1 | 1 | 1 | 1 | 1 | |
Tn5(uL) | 9 | 9 | 9 | 9 | 9 | 9 | |
Final vol | 10 | 10 | 10 | 10 | 10 | 10 | |
Final Conc.(uM) | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 |
A | B | |
Stock | Vol per 25uL rxn (uL) | |
10X Tango RE Buffer | 2.5 | |
Nuclease-free water | 18 | |
DMF | 2.5 | |
Linearized pUC19(25ng/uL) | 1 | |
Tn5 | 1 |
Stock | Vol for 10mL | Final Conc. | Supplier | |
1M Tris-HCl pH7-8 | 200uL | 20mM | Invitrogen #AM9850G, #AM9855G | |
1M MgCl2 | 100uL | 10mM | Invitrogen #AM9530G | |
Nuclease-free water | 9.7mL | Invitrogen AM9932 |
Primer pair:610 | ||||||
Sample | Cp | Ave Cp | delta Cp | |||
0.5 | 8.89 | 8.78 | 8.98 | 8.88 | 0.74 | |
0.5 | 9.12 | 8.81 | 8.97 | 8.97 | 0.83 | |
1 | 9.74 | 9.8 | 9.74 | 9.76 | 1.62 | |
1 | 9.67 | 9.99 | 9.79 | 9.82 | 1.68 | |
2.2 | 11.36 | 11.34 | 11.49 | 11.40 | 3.26 | |
2.2 | 11.37 | 11.67 | 11.57 | 11.54 | 3.40 | |
3.5 | 12.52 | 12.63 | 12.73 | 12.63 | 4.49 | |
3.5 | 12.7 | 12.74 | 12.86 | 12.77 | 4.63 | |
5 | 9.83 | 9.94 | 9.92 | 9.90 | 1.76 | |
5 | 9.79 | 10.05 | 9.97 | 9.94 | 1.80 | |
noTn5 | 8.2 | 8.02 | 8.18 | 8.13 | -0.01 | |
noTn5 | 8.25 | 7.97 | 8.21 | 8.14 | 0.00 | |
NTC | 24.04 | 23.92 | 23.9 | 23.95 | 15.81 | |
NTC | 24.27 | 24.25 | 24.26 | 24.26 | 16.12 |
Primer pair:610 | |||
Conc.(uM) | ave delta CP | stdev | |
0.5uM | 0.79 | 0.06 | |
1uM | 1.65 | 0.04 | |
2.2uM | 3.33 | 0.10 | |
3.5uM | 4.56 | 0.10 | |
5uM | 1.78 | 0.03 |