Oligos | Concentration | Working concentration | Synthesis scale | Purification | Supplier | Resuspension buffer | Sequence 5'-3' | |
IS1 | 500 uM | na | 100 nmol | HPLC | IDT | 10 mM Tris-HCl | A*C*A*C*TCTTTCCCTACACGACGCTCTTCCG*A*T*C*T | |
IS2 | 500 uM | na | 100 nmol | HPLC | IDT | TE | G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCG*A*T*C*T | |
IS3 | 500 uM | na | 100 nmol | HPLC | IDT | TE | A*G*A*T*CGGAA*G*A*G*C/3SpC3/ | |
CL104_duplex (order as double-stranded DNA) | 100 uM | 0.1 uM | 100 nmol | HPLC | IDT | TET buffer | /5Phos/TCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGA/3Phos/ | |
CL107 | 100 uM | 10 uM | 25 nmol | desalted | IDT | Water | TCATGTAACTCGCCTTGATCGT | |
CL108 | 100 uM | 10 uM | 25 nmol | desalted | IDT | Water | TCGTCGTTTGGTATGGCTTC | |
IS7 | 100 uM | 10 uM | 25 nmol | desalted | IDT | Water | ACACTCTTTCCCTACACGAC | |
IS8 | 100 uM | 10 uM | 25 nmol | desalted | IDT | Water | GTGACTGGAGTTCAGACGTGT | |
CL72_Custom_Seq_Primer | 100 uM | na | 25 nmol | desalted | IDT | Water | ACACTCTTTCCCTACACGACGCTCTTCC | |
CL72_i5_index | 100 uM | na | 25 nmol | desalted | IDT | Water | GGA AGA GCG TCG TGT AGG GAA AGA GTG T | |
CL105_CL106_Std | 100 uM | Dilute to 10^11 - 10^2 copies/ul | 4 nmol Ultramer | desalted | IDT | TET buffer | ACACTCTTTCCCTACACGACGCTCTTCCTCGTCGTTTGGTATGGCTTCTATCGUATCGATCGATCGACGATCAAGGCGAGTTACATGAAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC | |
P5-indexing primer | 100 uM | 10 uM | 25 nmol | desalted | IDT | Water | AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTT | |
P7-indexing primer | 100 uM | 10 uM | 25 nmol | desalted | IDT | Water | CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGT |
A | B | C | |
Tube | Qty | For ... | |
1.5 ml Safelock Tube | 10 | 5X SYBR | |
0.5 ml Safelock Tube | 8 | 25 mM dNTPs | |
1.5 ml Safelock Tube | 1 | 0.1 uM CL104_duplex | |
1.5 ml Safelock Tube | 1 | 0.1 uM CL105_duplex 1/500 | |
1.5 ml Safelock Tube | 1 | 10 uM IS7 | |
1.5 ml Safelock Tube | 1 | 10 uM IS8 | |
1.5 ml Safelock Tube | 1 | 10 uM CL107 | |
1.5 ml Safelock Tube | 1 | 10 uM CL108 | |
1.5 ml Safelock Tube | 10 | CL105_106 STD dilution series 10^11 - 10^2 | |
15 ml Falcon Tube | 1 | TE Buffer | |
15 ml Falcon Tube | 1 | TET buffer | |
1.5 ml Safelock Tube | 1 | Oligo hybridisation buffer | |
1.5 ml Safelock Tube | 1 | Reaction enhancer | |
15 ml Falcon Tube | 1 | EBT buffer | |
0.2 ml Lo-bind PCR tube | 2 | Adapters (P5_DS1, P7_DS2) | |
0.5 ml Lo-bind PCR tube | 1 | Adapters mix (DS_adapter_mix) | |
0.5 ml Lo-bind PCR tube | 1 | Adapters mix dilution | |
0.2 ml Lo-bind PCR Tube | # of samples + 2 | Reaction tubes | |
1.5 ml Lo-bind Tube | 3 | Step 19, Step 24, Step 27 master mixes | |
1.5 ml Lo-bind Tube | # of samples + 2 | Combining library with PB buffer | |
1.5 ml Lo-bind Tube | # of samples + 2 | Elution of library from spin column | |
QIAGEN MinElute PCR Purification Spin Columns | # of samples + 2 | Purification of library | |
0.5 ml Lo-bind Tube | # of samples + 2 | Final library | |
0.5 ml Lo-bind Tube | # of samples + 2 | 1/20 dilution of library | |
1.5 ml Safelock Tube | 2 | Assay A and B master mixes | |
8-strip optical qPCR Tubes | (((# of samples + 2)*2)+26)/8 | Assay A and B |
A | B | C | D | |
Buffer | Reagent | Volume to add | Final concentration in solution | |
Oligo hybridisation buffer 10X | 5 M NaCl | 100 ul | 500 mM | |
1 M Tris-HCl | 10 ul | 10 mM | ||
0.5 M EDTA | 2 ul | 1 mM | ||
Ultrapure water | 888 ul | na | ||
Reaction enhancer | 50% PEG-4000 | 500 ul | 25% | |
10 mg/ml BSA | 200 ul | 2 mg/ml | ||
5 M NaCl | 80 ul | 400 mM | ||
Ultrapure water | 220 ul | na | ||
TE Buffer | Ultrapure water | 9.88 ml | na | |
(Exp. 1 year) | 1 M Tris-HCl | 100 ul | 0.01 M | |
0.5 M EDTA | 20 ul | 0.001 M | ||
EBT | 1 M Tris-HCl | 100 ul | 0.01 M | |
100% Tween 20 | 5 ul | 0.05% | ||
Ultrapure water | 9.895 ml | na | ||
TET buffer | 1 M Tris-HCl | 500 ul | 0.01 M | |
(Exp. 1 year) | 0.5 M EDTA | 100 ul | 0.001 M | |
100% Tween 20 | 25 ul | 0.05% | ||
Ultrapure water | 49.375 ml | na | ||
5X SYBR | 10,000X SYBR | 2.5 ul | 5X | |
DMSO | 997.5 ul | na | ||
DMSO | 4 ml | na | ||
25 mM dNTPs | 100 mM dATP | 100 ul | 25 mM | |
100 mM dTTP | 100 ul | 25 mM | ||
100 mM dCTP | 100 ul | 25 mM | ||
100 mM dGTP | 100 ul | 25 mM |
A | B | C | |
Working stock | Reagent | Volume to add | |
10 uM CL104_duplex | 100 uM CL104 | 50 ul | |
TET buffer | 450 ul | ||
0.1 uM CL104_duplex | 10 uM CL104 | 5 ul | |
TET buffer | 495 ul | ||
0.1 uM CL104_duplex 1/500 | 0.1 uM CL104 | 1 ul | |
(i.e., 0.0002 uM) | TET buffer | 499 ul | |
10 uM IS7 | 100 uM IS7 | 50 ul | |
Ultrapure water | 450 ul | ||
10 uM IS8 | 100 uM IS8 | 50 ul | |
Ultrapure water | 450 ul | ||
10 uM CL107 | 100 uM CL107 | 50 ul | |
Ultrapure water | 450 ul | ||
10 uM CL108 | 100 uM CL108 | 50 ul | |
Ultrapure water | 450 ul | ||
10 uM CL105_106_STD | 100 uM CL105_106_STD | 50 ul | |
TET buffer | 450 ul | ||
10^11 copies CL105_106_STD | 10 uM CL105_106_STD | 10 ul | |
TET buffer | 592.25 ul | ||
10^10 copies CL105_106_STD | 10^11 copies CL105_106_STD | 50 ul | |
TET buffer | 450 ul | ||
10^9 copies CL105_106_STD | 10^10 copies CL105_106_STD | 50 ul | |
TET buffer | 450 ul | ||
10^8 copies CL105_106_STD | 10^9 copies CL105_106_STD | 50 ul | |
TET buffer | 450 ul | ||
10^7copies CL105_106_STD | 10^8 copies CL105_106_STD | 50 ul | |
TET buffer | 450 ul | ||
10^6 copies CL105_106_STD | 10^7copies CL105_106_STD | 50 ul | |
TET buffer | 450 ul | ||
10^5 copies CL105_106_STD | 10^6 copies CL105_106_STD | 50 ul | |
TET buffer | 450 ul | ||
10^4 copies CL105_106_STD | 10^5 copies CL105_106_STD | 50 ul | |
TET buffer | 450 ul | ||
10^3 copies CL105_106_STD | 10^4 copies CL105_106_STD | 50 ul | |
TET buffer | 450 ul | ||
10^2 copies CL105_106_STD | 10^3 copies CL105_106_STD | 50 ul | |
TET buffer | 450 ul |
2% Agarose Gel Electrophoresis
A | B | C | D | E | F | |
Reagent | V2 (reaction volume) | C1 (stock concentration) | C2 (concentration in reaction) | V1 (volume to add) | x _______ rxn | |
T4 DNA ligase buffer w/ 10 mM ATP (NEB) | 40 ul | 10 X | 1 X | 4 ul | ||
T4 DNA Polymerase | 40 ul | 3 U/ul | 0.03 U/ul | 0.4 ul | ||
T4 PNK | 40 ul | 10 U/ul | 0.25 U/ul | 1 ul | ||
Reaction enhancer | 40 ul | na | na | 2.2 ul | ||
dNTPs | 40 ul | 25 mM | 0.25 mM | 0.4 ul |
A | B | C | D | E | F | |
Reagent | V2 (reaction volume) | C1 (stock concentration) | C2 (concentration in reaction) | V1 (volume to add) | x _______ rxn | |
T4 DNA ligase buffer w/ 10 mM ATP (NEB) | 50 ul | 10 X | 0.2 X | 1 ul | ||
PEG-4000 | 50 ul | 50% | 6% | 6 ul | ||
T4 DNA ligase | 50 ul | 400 U/ul | 8 U/ul | 1 ul | ||
Ultrapure water | 50 ul | na | na | 1 ul |
A | B | C | D | E | F | |
Reagent | V2 (reaction volume) | C1 (stock concentration) | C2 (concentration in reaction) | V1 (volume to add) | x _______ rxn | |
Isothermal amplification buffer | 60 ul | 10 X | 0.33 X | 2 ul | ||
dNTPs | 60 ul | 25 mM | 0.33 mM | 0.8 ul | ||
Bst 2.0 Warmstart DNA polymerase | 60 ul | 8 U/ul | 0.213 U/ul | 1.6 ul | ||
Ultrapure water | 60 ul | na | na | 5.6 ul |
A | B | C | D | E | F | |
Reagent | V2 | C1 | C2 | V1 | x _______ rxn | |
Ultrapure water | 25 ul | na | na | 15.9 ul | ||
BSA | 25 ul | 10 mg /ml | 0.4 mg/ml | 1 ul | ||
ABI Gold PCR Buffer | 25 ul | 10 X | 1 X | 2.5 ul | ||
MgCl2 | 25 ul | 25 mM | 2.5 mM | 2.5 ul | ||
dNTPs | 25 ul | 25 mM | 0.25 mM | 0.25 ul | ||
ABI Taq Gold DNA polymerase | 25 ul | 5 U/ul | 0.05 U/ul | 0.25 ul | ||
SYBR Green | 25 ul | 5 X | 0.12 X | 0.6 ul | ||
IS7 | 25 ul | 10 uM | 0.2 uM | 0.5 ul | ||
IS8 | 25 ul | 10 uM | 0.2 uM | 0.5 ul |
A | B | C | D | E | F | |
Reagent | V2 | C1 | C2 | V1 | x __16___ rxn | |
Ultrapure water | 25 ul | na | na | 15.9 ul | 254.4 ul | |
BSA | 25 ul | 10 mg /ml | 0.4 mg/ml | 1 ul | 16 ul | |
ABI Gold PCR Buffer | 25 ul | 10 X | 1 X | 2.5 ul | 40 ul | |
MgCl2 | 25 ul | 25 mM | 2.5 mM | 2.5 ul | 40 ul | |
dNTPs | 25 ul | 25 mM | 0.25 mM | 0.25 ul | 4 ul | |
ABI Taq Gold DNA polymerase | 25 ul | 5 U/ul | 0.05 U/ul | 0.25 ul | 4 ul | |
SYBR Green | 25 ul | 5 X | 0.12 X | 0.6 ul | 9.6 ul | |
CL107 | 25 ul | 10 uM | 0.2 uM | 0.5 ul | 8 ul | |
CL108 | 25 ul | 10 uM | 0.2 uM | 0.5 ul | 8 ul |
A | B | C | D | |
PCR NTC | CL105_106_STD 10^3 | dsLib001 Neat | dsLib005 Neat | |
PCR NTC | CL105_106_STD 10^3 | dsLib001 1in20 | dsLib005 1in20 | |
CL105_106_STD 10^6 | CL105_106_STD 10^2 | dsLib002 Neat | ...etc. | |
CL105_106_STD 10^6 | CL105_106_STD 10^2 | dsLib002 1in20 | ||
CL105_106_STD 10^5 | dsCL104 +VE Neat | dsLib003 Neat | ||
CL105_106_STD 10^5 | dsCL104 +VE 1in20 | dsLib003 1in20 | ||
CL105_106_STD 10^4 | dsNTC -VE Neat | dsLib004 Neat | ||
CL105_106_STD 10^4 | dsNTC -VE 1in20 | dsLib004 1in20 |
A | B | |
0.1 uM CL104_duplex 1/500 | CL105_106_STD 10^4 | |
0.1 uM CL104_duplex 1/500 | CL105_106_STD 10^4 | |
PCR NTC | CL105_106_STD 10^3 | |
PCR NTC | CL105_106_STD 10^3 | |
CL105_106_STD 10^6 | CL105_106_STD 10^2 | |
CL105_106_STD 10^6 | CL105_106_STD 10^2 | |
CL105_106_STD 10^5 | ||
CL105_106_STD 10^5 |
2% Agarose Gel Electrophoresis
A | B | C | D | E | F | |
Reagent | V2 | C1 | C2 | V1 | x _____ rxn | |
Ultrapure water | 25 ul | na | na | 10.9 | ||
BSA | 25 ul | 10 mg /ml | 0.4 mg/ml | 1 ul | ||
ABI Gold PCR Buffer | 25 ul | 10 X | 1 X | 2.5 ul | ||
MgCl2 | 25 ul | 25 mM | 2.5 mM | 2.5 ul | ||
dNTPs | 25 ul | 25 mM | 0.25 mM | 0.25 ul | ||
ABI Taq Gold DNA polymerase | 25 ul | 5 U/ul | 0.05 U/ul | 0.25 ul | ||
SYBR Green | 25 ul | 5 X | 0.12 X | 0.6 ul | ||
P5_indexing_primer | 25 ul | 10 uM | 0.2 uM | 0.5 ul | Don't add to master mix | |
P7_indexing_primer | 25 ul | 10 uM | 0.2 uM | 0.5 ul | Don't add to master mix |
A | B | C | D | E | F | |
dsLib001 | dsLib003 | dsLib005 | ||||
dsLib001 | dsLib003 | dsLib005 | ||||
dsLib001 | dsLib003 | dsLib005 | ||||
dsLib001 | dsLib003 | dsLib005 | ||||
dsLib002 | dsLib004 | ...etc. | ||||
dsLib002 | dsLib004 | |||||
dsLib002 | dsLib004 | |||||
dsLib002 | dsLib004 |
Tube | Qty | For ... | |
1.5 ml Lo-bind Safelock tube | 1 | 10 mM Tris-HCl | |
1.5 ml Lo-bind Safelock tube | 1 | 10X Annealing Buffer | |
0.5 ml Lo-bind Safelock tube | 2 | P1 and P2 adapter oligos | |
0.2 ml Lo-bind PCR tube | 1 | Annealing P1 and P2 adapter oligos |
Buffer | Reagent | Volume to add | Final concentration in solution | |
10 mM Tris-HCl | 1 M Tris-HCl | 10 ul | 10 mM | |
Ultrapure water | 990 ul | na | ||
10 X Annealing Buffer | 1 M Tris-HCl | 100 ul | 100 mM | |
5 M NaCl | 100 ul | 500 mM | ||
0.5 M EDTA | 20 ul | 10 mM | ||
Ultrapure water | 780 ul | na |
Working stock | Reagent | Volume to add | |
10 uM P5_Indexing_Primer | 100 uM Stock | 50 ul | |
Ultrapure water | 450 ul | ||
10 uM P7_Indexing_Primer | 100 uM Stock | 50 ul | |
Ultrapure water | 450 ul |
Reagent | V2 (reaction volume) | C1 (stock concentration) | C2 (concentration in reaction) | V1 (volume to add) | |
P1.1 adapter oligo (EcoRI) | 100 ul | 100 uM | 10 uM | 10 ul | |
P1.2 adapter oligo (EcoRI) | 100 ul | 100 uM | 10 uM | 10 ul | |
Annealing buffer | 100 ul | 10 X | 1 X | 10 ul | |
Ultrapure water | 100 ul | na | na | 70 ul |
Reagent | V2 (reaction volume) | C1 (stock concentration) | C2 (concentration in reaction) | V1 (volume to add) | |
P2.1 adapter oligo (MspI) | 100 ul | 100 uM | 10 uM | 10 ul | |
P2.2 adapter oligo (MspI) | 100 ul | 100 uM | 10 uM | 10 ul | |
Annealing buffer | 100 ul | 10 X | 1 X | 10 ul | |
Ultrapure water | 100 ul | na | na | 70 ul |
Tube | Qty | For ... | |
1.5 ml Lo-Bind Safelock tube | 1 | Step 2.2 Master mix | |
0.2 ml Lo-Bind PCR tube | # of samples | Enzymatic digestion for samples |
Reagent | V2 (reaction volume) | C1 (stock concentration) | C2 (concentration in reaction) | V1 (volume to add) | |
CutSmart Buffer (NEB) | 50 ul | 10 X | 1 X | 5 ul | |
EcoRI-HF (NEB) | 50 ul | 20 U/ul | 0.4 U/ul (20 U) | 1 ul | |
MspI (NEB) | 50 ul | 20 U/ul | 0.4 U/ul (20 U) | 1 ul | |
Ultrapure water | 50 ul | na | na | 33 ul |
2% Agarose Gel Electrophoresis
2% Agarose Gel Electrophoresis
Tube | Qty | For ... | |
1.5 ml Lo-Bind Safelock tube | 1 | Step 3.2 Master mix | |
0.2 ml Lo-Bind PCR tube | # of samples x 2 | Reactions, two per sample |
Reagent | V2 (reaction volume) | C1 (stock concentration) | C2 (concentration in reaction) | V1 (volume to add) | |
CutSmart Buffer (NEB) | 20 ul | 10 X | 1 X | 2 ul | |
P1 adapter | 20 ul | 10 uM | 0.5 uM | 1 ul | |
P2 adapter | 20 ul | 20 uM | 1 uM | 1 ul | |
ATP | 20 ul | 100 mM | 1 mM | 0.2 ul | |
T4 DNA ligase | 20 ul | 400 U/ul | 20 U/ul (400 U) | 1 ul | |
Ultrapure water | 20 ul | na | na | 5.8 ul |
Tube | Qty | For ... | |
1.5 ml Lo-Bind Safelock tube | 1 | Step 4.2 Master mix | |
8-well strip qPCR tubes 0.1 ul profile | 1 | PCR amplification |
Reagent | V2 | C1 | C2 | V1 | x _____ rxn | |
Ultrapure water | 25 ul | na | na | 15.9 ul | ||
BSA | 25 ul | 10 mg /ml | 0.4 mg/ml | 1 ul | ||
ABI Gold PCR Buffer | 25 ul | 10 X | 1 X | 2.5 ul | ||
MgCl2 | 25 ul | 25 mM | 2.5 mM | 2.5 ul | ||
dNTPs | 25 ul | 25 mM | 0.25 mM | 0.25 ul | ||
ABI Taq Gold DNA polymerase | 25 ul | 5 U/ul | 0.05 U/ul | 0.25 ul | ||
SYBR Green | 25 ul | 5 X | 0.12 X | 0.6 ul | ||
IS7 | 25 ul | 10 uM | 0.2 uM | 0.5 ul | ||
IS8 | 25 ul | 10 uM | 0.2 uM | 0.5 ul |
PCR NTC | ||||
PCR NTC | ||||
MD#033 Neat | ||||
MD#033 1 in 20 | ||||
MD#034 Neat | ||||
MD#034 1 in 20 | ||||
2% Agarose Gel Electrophoresis
Tube | Qty | For ... | |
1.5 ml Lo-Bind Safelock tube | 1 | Step 7.2 Master mix | |
8-well strip qPCR tubes 0.1 ul profile | 1 | PCR amplification |
Reagent | V2 | C1 | C2 | V1 | x _____ rxn | |
Ultrapure water | 50 ul | na | na | 29.5 ul | ||
KAPA High Fidelity Buffer | 50 ul | 5 X | 1 X | 10 ul | ||
P5_indexing_primer | 50 ul | 5 uM | 0.2 uM | 2 ul | Do not add to master mix | |
P7_indexing_primer | 50 ul | 5 uM | 0.2 uM | 2 ul | Do not add to master mix | |
KAPA HiFi Hot Start DNA Polymerase | 50 ul | 1 U/ul | 0.02 U/ul | 1 ul | ||
dNTPs | 50 ul | 25 uM | 0.25 uM | 0.5 ul |
MD#033 | ||||
MD#033 | ||||
MD#033 | ||||
MD#033 | ||||
MD#034 | ||||
MD#034 | ||||
MD#034 | ||||
MD#034 |
2% Agarose Gel Electrophoresis
Tube | Qty | For ... | |
1.5 ml Lo-Bind Safelock tube | 1 | Pooled RAD library | |
1.5 ml Lo-Bind Safelock tube | 1 | Aliquot of RAD library for sequencing |
Tube | Qty | For ... | |
1.5 ml Lo-Bind Safelock tube | 1 | Step 9.2 master mix | |
0.2 ml Lo-Bind PCR tube | 1 per 1 ug DNA | Adapter removal reaction |
Reagent | V2 (reaction volume) | C1 (stock concentration) | C2 (concentration in reaction) | V1 (volume to add) | x ______ rxn | |
CutSmart Buffer (NEB) | 50 ul | 10 X | 1 X | 5 ul | ||
MspI | 50 ul | 20 U/ul | 0.4 U/ul (20 U) | 1 ul | ||
Ultrapure water | 50 ul | na | na | 25.4 ul |
2% Agarose Gel Electrophoresis
Tube | Qty | For ... | |
1.5 ml Lo-Bind Safelock tube | 1 | Step 10.3 master mix | |
0.2 ml Lo-Bind PCR tube | # reactions | Transcription/biotinylation reaction | |
0.2 ml Lo-Bind PCR tube | 2 | Aliquots of TURBO DNase and SuperaseIn RNAse inhibitor | |
1.5 ml Lo-Bind Safelock tube | 1 | Combine probes for cleanup | |
RNeasy mini spin column | 1 | Purification | |
1.5 ml Lo-Bind Safelock tube with the lid cut off | 1 | Elution | |
1.5 ml Lo-Bind Safelock tube | 1 | Final tube for probes | |
0.5 ml Lo-Bind Safelock tube | Depends on total ug of probes | 6-ul aliquots of probes |
Reagent | V2 (reaction volume) | C1 (stock concentration) | C2 (concentration in reaction) | V1 (volume to add) | x ______ rxn | |
Reaction Buffer (HiScribe kit) | 20 ul | 10 X | 0.75 X | 1.5 ul | ||
dATP | 20 ul | 100 uM | 7.5 uM | 1.5 ul | ||
dCTP | 20 ul | 100 uM | 7.5 uM | 1.5 ul | ||
dGTP | 20 ul | 100 uM | 7.5 uM | 1.5 ul | ||
dUTP | 20 ul | 100 uM | 5 uM | 1 ul | ||
biotin-UTP | 20 ul | 10 mM | 2.5 mM | 5 ul | ||
T7 RNA polymerase mix | 20 ul | ? | ? | 1.5 ul | ||
Ultrapure water | 20 ul | na | na | Adjust depending on the volume of library added |
Tube | Qty | For ... | |
1.5 ml Lo-Bind Safelock tube | 1 | Mineral oil aliquot | |
0.2 ml Lo-Bind PCR tube | # capture reactions x 2 | Hybridisation | |
1.5 ml Lo-Bind Safelock tube | 2 | Step 14 and 15 master mixes |
Reagent | Stored at... | Thaw at... | |
20 X SSPE | 4 deg C | Room temperature | |
500 mM EDTA | 4 deg C | Room temperature | |
50 X Denardt's Solution | -20 deg C | Room temperature | |
10% SDS | 4 deg C | Room temperature | |
Chickent Cot-1 (HyBloc) | -20 deg C | On ice | |
FWD_blocking_primer | -20 deg C | On ice | |
REV_blocking_primer | -20 deg C | On ice | |
SUPERas-In RNAse inhibitor | -20 deg C | On ice | |
Salmon Sperm DNA | -20 deg C | On ice |
Reagent | V2 (reaction volume) | C1 (stock concentration) | C2 (concentration in reaction) | V1 (volume to add) | x ______ rxn | |
Chicken Cot-1 (HyBloc) | 3.25 ul | 1 ug/ul | 0.77 ug/ul | 2.5 ul | ||
Salmon Sperm DNA | 3.25 ul | 10 ug/ul | 0.77 ug/ul | 0.25 ul | ||
FWD_blocking_primer | 3.25 ul | 200 uM | 15.4 uM | 0.25 ul | ||
REV_blockg_primer | 3.25 ul | 200 uM | 15.4 uM | 0.25 ul |
Reagent | V2 (reaction volume) | C1 (stock concentration) | C2 (concentration in reaction) | V1 (volume to add) | x ______ rxn | |
SSPE | 20 ul | 20 X | 9 X | 9 ul | ||
EDTA | 20 ul | 0.5 M | 0.0125 M | 0.5 ul | ||
Denhardt's solution | 20 ul | 50 X | 8.75 X | 3.5 ul | ||
SDS | 20 ul | 10 % | 0.25 % | 0.5 ul | ||
SUPERase-In RNase inhibitor | 20 ul | 20 U/ul | 1 U/ul | 1 ul |
Tube | Qty | For ... | |
50 ml Falcon tubes | 3 | Wash buffer 2, Wash buffer 2.2, Binding buffer | |
15 ml Falcon tubes | 3 | Aliquots for Wash buffer 2.2 and Binding buffer, and 10 mM Tris-HCl/0.05% Tween-20 | |
1.5 ml Lo-bind Safelock tube | 1 | Aliquot for MyOne C1 Streptavidin beads | |
1.5 ml Lo-bind Safelock tube | 3 x # hybridisation reactions | Enrichment |
Buffer | Reagent | Volume | C2 | |
Wash buffer 2 | 20 X SSC | 100 ul | 0.1 X | |
10% SDS | 200 ul | 0.1 % | ||
Ultrapure water | 19.7 ml | na | ||
Wash buffer 2.2 | 10% SDS | 400 ul | 0.08% | |
Wash buffer 2 | 10 ml | na | ||
Ultrapure water | 39.6 ml | na | ||
Binding buffer | 5 M NaCl | 10 ml | 1 M | |
1 M Tris-HCl | 500 ul | 10 mM | ||
0.5 M EDTA | 100 ul | 1 mM | ||
Ultrapure water | 39.4 ml | na | ||
10 mM Tris-HCl, 0.05% Tween-20 | 1 M Tris-HCl | 500 ul | 10 mM | |
100% Tween-20 | 25 ul | 0.05% | ||
Ultrapure water | 49.475 ml | na |
700 ul * # reactions | Binding buffer | |
30 ul * # reactions | MyOne C1 Streptavidin beads | |
35 ul * # reactions | 10 mM Tris-HCl/0.05% Tween-20 | |
1600 ul * # reactions | Wash Buffer 2.2 |
Oligo | Reagent | Volume | |
10 uM P5_primer | 100 uM P5 | 50 ul | |
Ultrapure water | 450 ul | ||
10 uM P7_primer | 100 uM P7 | 50 ul | |
Ultrapure water | 450 ul |
Reagent | V2 | C1 | C2 | V1 | x _____ rxn | |
Ultrapure water | 25 ul | na | na | 10.9 | ||
BSA | 25 ul | 10 mg /ml | 0.4 mg/ml | 1 ul | ||
ABI Gold PCR Buffer | 25 ul | 10 X | 1 X | 2.5 ul | ||
MgCl2 | 25 ul | 25 mM | 2.5 mM | 2.5 ul | ||
dNTPs | 25 ul | 25 mM | 0.25 mM | 0.25 ul | ||
ABI Taq Gold DNA polymerase | 25 ul | 5 U/ul | 0.05 U/ul | 0.25 ul | ||
SYBR Green | 25 ul | 5 X | 0.12 X | 0.6 ul | ||
P5 | 25 ul | 10 uM | 0.4 uM | 1 ul | ||
P7 | 25 ul | 10 uM | 0.4 uM | 1 ul |
Capture001 Neat | ||
Capture001 1in10 | ||
Capture002 Neat | ||
Capture002 1in10 | ||
Capture003 Neat | ||
Capture003 1in10 | ||
PCR NTC | ||
PCR NTC |
Reagent | V2 | C1 | C2 | V1 | x _____ rxn | |
Ultrapure water | 25 ul | na | na | 10.9 | ||
BSA | 25 ul | 10 mg /ml | 0.4 mg/ml | 1 ul | ||
ABI Gold PCR Buffer | 25 ul | 10 X | 1 X | 2.5 ul | ||
MgCl2 | 25 ul | 25 mM | 2.5 mM | 2.5 ul | ||
dNTPs | 25 ul | 25 mM | 0.25 mM | 0.25 ul | ||
ABI Taq Gold DNA polymerase | 25 ul | 5 U/ul | 0.05 U/ul | 0.25 ul | ||
SYBR Green | 25 ul | 5 X | 0.12 X | 0.6 ul | ||
P5 | 25 ul | 10 uM | 0.4 uM | 1 ul | ||
P7 | 25 ul | 10 uM | 0.4 uM | 1 ul |
Capture001 Neat | Capture003 Neat | |
Capture001 Neat | Capture003 Neat | |
Capture001 Neat | Capture003 Neat | |
Capture001 Neat | Capture003 Neat | |
Capture002 Neat | ||
Capture002 Neat | ||
Capture002 Neat | ||
Capture002 Neat |
2% Agarose Gel Electrophoresis