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This page contains the protocols for the experiments throughout our project. For more information about each protocol, please refer to Engineering Success and Results.

Protocol Entries

[P01]

See experiment E1.1
See Development of Robust ssDNA Circularisation Protocol

  1. Into a PCR tube, add:
Sample 1
RCA linear template (v1) (100 µM stock) 2 μL
Linear scaffold DNA (v1) (100 µM stock) 2 μL
ddH₂O 13 μL
TOTAL 17 μL
  1. Incubate the reaction mixture at 95°C for 5 minutes; set the thermocycler to gradually cool down to 25°C.
  2. Into the same PCR tube, add:
Sample 1
Reaction mixture from step 1 17 μL
10× T4 Ligase Buffer 2 μL
T4 Ligase 1 μL
TOTAL 20 μL
  1. Incubate at 22°C for 15 minutes.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 17 μL ddH₂O preheated at 65°C.
  3. Into a new PCR tube, add:
Sample 1
Oligonucleotide purification product 17 μL
10× Exonuclease I reaction buffer 2 μL
Exonuclease I 1 μL
TOTAL 20 μL
  1. Incubate at 37°C for 2 hours.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 20 μL ddH₂O preheated at 65°C.
  3. Measure the sample concentration and dilute to 1 ng/μL.

[P02]

See experiment E1.1

  1. Into the PCR tube, add:
(1) (+) With IDT primer (2) (-) Without primer
ddH₂O 12 μL 14 μL
rCutSmart Buffer 2 μL 2 μL
dNTP mix (10 mM each, stock) 2 μL 2 μL
RCA template (v1) (1 ng/μL stock) 1 μL 1 μL
RCA primer (10 µM stock) 2 μL -
TOTAL 19 μL 19 μL
  1. Incubate at 95°C for 5 minutes, and then return to ice.
  2. Add 1 μL of Φ29 DNA polymerase to each tube.
  3. Incubate at 30°C for 2 hours.
  4. Perform heat inactivation at 65°C for 10 minutes.
  5. Run 10 μL of each sample to gel electrophoresis using 0.8% Agarose/TAE gel with RedSafe.

[P03]

See experiment E1.1

  1. Into each PCR tube, add:
(1) (2) (-) control (+) control
10× G-quadruplex buffer 12.5 μL 12.5 μL 12.5 μL 12.5 μL
G-quadruplex sample 10 μL from (1) (+) With IDT primer 10 μL from (2) (-) Without primer 10 μL from failed RCA 6.25 μL from IDT G-quad 8 µM stock
Haemin, in DMSO (1 mM stock) 1 μL 1 μL 1 μL 1 μL
ddH₂O 98.5 μL 98.5 μL 98.5 μL 102.25 μL
TMB (50 mM stock in DMSO) 1.5 μL 1.5 μL 1.5 μL 1.5 μL
H₂O₂ 1.5 μL 1.5 μL 1.5 μL 1.5 μL
TOTAL 125 μL 125 μL 125 μL 125 μL

[P04]

See experiment E1.2

  1. Into a PCR tube, add:
Sample 1
RCA linear template (v2) (100 µM stock) 2 μL
Linear scaffold DNA (v2) (100 µM stock) 2 μL
ddH₂O 13 μL
TOTAL 17 μL
  1. Incubate the reaction mixture at 95°C for 5 minutes; set the thermocycler to gradually cool down to 25°C.
  2. Into the same PCR tube, add:
Sample 1
Reaction mixture from step 1 17 μL
10× T4 Ligase Buffer 2 μL
T4 Ligase 1 μL
TOTAL 20 μL
  1. Incubate at 22°C for 15 minutes.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 17 μL ddH₂O preheated at 65°C.
  3. Into a new PCR tube, add:
Sample 1
Oligonucleotide purification product 17 μL
10× Exonuclease I reaction buffer 2 μL
Exonuclease I 1 μL
TOTAL 20 μL
  1. Incubate at 37°C for 2 hours.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 20 μL ddH₂O preheated at 65°C.
  3. Measure the sample concentration and dilute to 1 ng/μL.

[P05]

See experiment E1.2

  1. Into the PCR tubes, add:
(1) (+) With IDT primer (2) (-) Without primer
ddH₂O 12 μL 14 μL
rCutSmart Buffer 2 μL 2 μL
dNTP mix (10 mM each, stock) 2 μL 2 μL
RCA template (v2) (1 ng/μL stock) 1 μL 1 μL
RCA primer (10 µM stock) 2 μL -
TOTAL 19 μL 19 μL
  1. Incubate at 95°C for 5 minutes, and then return to ice.
  2. Add 1 μL of Φ29 DNA polymerase to each tube.
  3. Incubate at 30°C for 2 hours.
  4. Perform heat inactivation at 65°C for 10 minutes.
  5. Run 10 μL of each sample to gel electrophoresis using 0.8% Agarose/TAE gel with RedSafe.

[P06]

See experiment E1.3

  1. Into a PCR tube, add:
Sample 1
RCA linear template (v3) (100 µM stock) 2 μL
Linear scaffold DNA (v3) (100 µM stock) 2 μL
ddH₂O 13 μL
TOTAL 17 μL
  1. Incubate the reaction mixture at 95°C for 5 minutes; set the thermocycler to gradually cool down to 25°C.
  2. Into the same PCR tube, add:
Sample 1
Reaction mixture from step 1 17 μL
10× T4 Ligase Buffer 2 μL
T4 Ligase 1 μL
TOTAL 20 μL
  1. Incubate at 22°C for 15 minutes.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 17 μL ddH₂O preheated at 65°C.
  3. Into a new PCR tube, add:
Sample 1
Oligonucleotide purification product 16.8 μL
10× Exonuclease I reaction buffer 2 μL
Exonuclease I 1 μL
Triton X-100 0.2 μL
TOTAL 20 μL
  1. Incubate at 37°C for 2 hours.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 20 μL ddH₂O preheated at 65°C.
  3. Measure the sample concentration and dilute to 1 ng/μL.

[P07]

See experiment E1.3

  1. Into the PCR tubes, add:
(1) (+) Triton & ExoI-treated, with IDT primer (2) (-) Triton & ExoI-treated, without primer (3) (+) ExoI-treated, with IDT primer (4) (-) ExoI-treated, without primer
ddH₂O 12 μL 14 μL 12 μL 14 μL
rCutSmart Buffer 2 μL 2 μL 2 μL 2 μL
dNTP mix (10 mM each, stock) 2 μL 2 μL 2 μL 2 μL
RCA template (v3) (1 ng/μL stock) 1 μL (Triton-100 and ExoI-treated) 1 μL (Triton-100 and ExoI-treated) 1 μL (ExoI-treated) 1 μL (ExoI-treated)
RCA primer (10 µM stock) 2 μL - 2 μL -
TOTAL 19 μL 19 μL 19 μL 19 μL
  1. Incubate at 95°C for 5 minutes, and then return to ice.
  2. Add 1 μL of Φ29 DNA polymerase to each tube.
  3. Incubate at 30°C for 2 hours.
  4. Perform heat inactivation at 65°C for 10 minutes.
  5. Run 10 μL of each sample to gel electrophoresis using 0.8% Agarose/TAE gel with GelGreen.

[P08]

See experiment E1.4
See Development of Robust ssDNA Circularisation Protocol

  1. Into a PCR tube, add:
Sample 1
RCA linear template (v3) (100 µM stock) 2 μL
Linear scaffold DNA (v3) (100 µM stock) 2 μL
ddH₂O 13 μL
TOTAL 17 μL
  1. Incubate the reaction mixture at 95°C for 5 minutes; set the thermocycler to gradually cool down to 25°C.
  2. Into the same PCR tube, add:
Sample 1
Reaction mixture from step 1 17 μL
10× T4 Ligase Buffer 2 μL
T4 Ligase 1 μL
TOTAL 20 μL
  1. Incubate at 22°C for 15 minutes.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 18 μL ddH₂O preheated at 65°C.
  3. Into a new PCR tube, add:
Sample 1
ddH₂O 70.5 μL
Oligonucleotide purification product 18 μL
10× Exonuclease III buffer 10 μL
Exonuclease III (TaKaRa Bio) 0.5 μL
Exonuclease I (Thermo Fisher Scientific) 1 μL
TOTAL 100 μL
  1. Incubate at 37°C for 1 hour.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 20 μL ddH₂O preheated at 65°C.
  3. Measure the sample concentration and dilute to 1 ng/μL.

[P09]

See experiment E1.4

  1. Into the PCR tubes, add:
(1) (+) ExoI & ExoIII-treated, with IDT primer, cut with Nt.BsmAI (2) (+) ExoI & ExoIII-treated, with IDT primer (3) (-) ExoI & ExoIII-treated, without primer
ddH₂O 11.5 μL 12 μL 14 μL
rCutSmart Buffer 2 μL 2 μL 2 μL
dNTP mix (2.5 mM each, stock) 2 μL 2 μL 2 μL
RCA template (v3) (1 ng/μL stock) 1 μL 1 μL 1 μL
RCA primer (10 µM stock) 2 μL 2 μL -
TOTAL 18.5 μL 19 μL 19 μL
  1. Incubate at 95°C for 5 minutes, and then return to ice.
  2. Into the same PCR tubes, add:
(1) (+) ExoI & ExoIII-treated, with IDT primer, cut with Nt.BsmAI (2) (+) ExoI & ExoIII-treated, with IDT primer (3) (-) ExoI & ExoIII-treated, without primer
Product from previous steps 18.5 μL 19 μL 19 μL
Φ29 DNA polymerase 1 μL 1 μL 1 μL
Nt.BsmAI 0.5 μL - -
TOTAL 20 μL 20 μL 20 μL
  1. Incubate at 37°C for 2 hours.
  2. Perform heat inactivation at 65°C for 20 minutes.
  3. Run 10 μL of each sample to gel electrophoresis using 0.8% Agarose/TAE gel with GelGreen.

[P10]

See experiment E1.4

  1. Into each PCR tube, add:
(1) (+) ExoI & ExoIII-treated, with IDT primer, cut with Nt.BsmAI (2) (+) ExoI & ExoIII-treated, with IDT primer (3) (-) ExoI & ExoIII-treated, without primer (+) control, from IDT G-quad 8 µM stock
10× G-quadruplex buffer 12.5 μL 12.5 μL 12.5 μL 12.5 μL
G-quadruplex sample 10 μL 10 μL 10 μL 6.25 μL
Haemin, in DMSO (1 mM stock) 1 μL 1 μL 1 μL 1 μL
ddH₂O 98.5 μL 98.5 μL 98.5 μL 102.25 μL
TMB (50 mM stock in DMSO) 1.5 μL 1.5 μL 1.5 μL 1.5 μL
H₂O₂ 1.5 μL 1.5 μL 1.5 μL 1.5 μL
TOTAL 125 μL 125 μL 125 μL 125 μL

[P11]

See experiment E2.1

  1. Into each centrifuge tube, add:
Sample 1
DNA-cleaving DNAzymes 1 and 2 2 µM each
10× DNA-cleaving DNAzyme buffer 5 μL
ddH₂O top up until (50 μL - test fragment)
TOTAL (50 μL - test fragment)
  1. Incubate at 95°C for 5 minutes, followed by gradual cooling down to 25°C over 15 minutes.
  2. Into the centrifuge tube, add:
Sample 1
Products from step 2 (50 μL - test fragment)
Test fragment (simulating RCA products) 5 nM
TOTAL 50 μL
  1. Incubate at room temperature for 40 minutes.
  2. Run the sample to gel electrophoresis using 5% Agarose/TAE gel with GelGreen.

[P12]

See experiment E2.2

  1. Into a PCR tube, add:
Sample 1
RCA linear template (v1) (100 µM stock) 2 μL
Linear scaffold DNA (v1) (100 µM stock) 2 μL
ddH₂O 13 μL
TOTAL 17 μL
  1. Incubate the reaction mixture at 95°C for 5 minutes; set the thermocycler to gradually cool down to 25°C.
  2. Into the same PCR tube, add:
Sample 1
Reaction mixture from step 1 17 μL
10× T4 Ligase Buffer 2 μL
T4 Ligase 1 μL
TOTAL 20 μL
  1. Incubate at 22°C for 15 minutes.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 17 μL ddH₂O preheated at 65°C.
  3. Into a new PCR tube, add:
Sample 1
Oligonucleotide purification product 17 μL
10× Exonuclease I reaction buffer 2 μL
Exonuclease I 1 μL
TOTAL 20 μL
  1. Incubate at 37°C for 2 hours.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 20 μL ddH₂O preheated at 65°C.
  3. Measure the sample concentration and dilute to 1 ng/μL.

[P13]

See experiment E2.2

  1. Into the PCR tube, add:
Sample 1
ddH₂O 11.4 μL
rCutSmart Buffer 2 μL
dNTP mix (10 mM each, stock) 2 μL
RCA template (v1) (1 ng/μL stock) 1 μL
RCA primer (10 µM stock) 2 μL
DTT (0.1 M stock) 0.6 μL
TOTAL 19 μL
  1. Incubate at 95°C for 5 minutes, and then return to ice.
  2. Add 1 μL of Φ29 DNA polymerase to each tube.
  3. Incubate at 30°C for 2 hours.
  4. Perform heat inactivation at 65°C for 10 minutes.
  5. Run 10 μL of the sample to gel electrophoresis using 0.8% Agarose/TAE gel with GelGreen.

[P14]

See experiment E2.2

  1. Into each PCR tube, add:
(+) RCA-generated G-quadruplex (+) IDT-synthesised G-quadruplex with DTT (+) IDT-synthesised G-quadruplex
10× G-quadruplex buffer 25 μL 25 μL 25 μL
G-quadruplex sample 10 μL 12.5 μL 12.5 μL
DTT (0.1 M stock) - 0.6 μL -
Haemin, in DMSO (1 mM stock) 2 μL 2 μL 2 μL
ddH₂O 207 μL 203.9 μL 204.5 μL
TMB (50 mM stock in DMSO) 3 μL 3 μL 3 μL
H₂O₂ 3 μL 3 μL 3 μL
TOTAL 250 μL 250 μL 250 μL

[P15]

See experiment E2.3

  1. Into a PCR tube, add:
Sample 1
RCA linear template (v1) (100 µM stock) 2 μL
Linear scaffold DNA (v1) (100 µM stock) 2 μL
ddH₂O 13 μL
TOTAL 17 μL
  1. Incubate the reaction mixture at 95°C for 5 minutes; set the thermocycler to gradually cool down to 25°C.
  2. Into the same PCR tube, add:
Sample 1
Reaction mixture from step 1 17 μL
10× T4 Ligase Buffer 2 μL
T4 Ligase 1 μL
TOTAL 20 μL
  1. Incubate at 22°C for 15 minutes.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 17 μL ddH₂O preheated at 65°C.
  3. Into a new PCR tube, add:
Sample 1
Oligonucleotide purification product 17 μL
10× Exonuclease I reaction buffer 2 μL
Exonuclease I 1 μL
TOTAL 20 μL
  1. Incubate at 37°C for 2 hours.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 20 μL ddH₂O preheated at 65°C.
  3. Measure the sample concentration and dilute to 1 ng/μL.

[P16]

See experiment E2.3

  1. Into the PCR tubes, add:
(1) (+) With IDT primer (2) (-) Without primer
ddH₂O 12 μL 14 μL
rCutSmart Buffer 2 μL 2 μL
dNTP mix (10 mM each, stock) 2 μL 2 μL
RCA template (v1) (1 ng/μL stock) 1 μL 1 μL
RCA primer (10 µM stock) 2 μL -
TOTAL 19 μL 19 μL
  1. Incubate at 95°C for 5 minutes, and then return to ice.
  2. Add 1 μL of Φ29 DNA polymerase to each tube.
  3. Incubate at 30°C for 2 hours.
  4. Perform heat inactivation at 65°C for 10 minutes.
  5. Run 10 μL of each sample to gel electrophoresis using 0.8% Agarose/TAE gel with GelGreen.

[P17]

See experiment E2.3

  1. Into each PCR tube, add:
(1) (+) RCA-generated G-quadruplex (2) (-) false positive RCA result
10× G-quadruplex buffer 12.5 μL 12.5 μL
G-quadruplex sample 10 μL from (1) (+) With IDT primer 10 μL from (2) (-) Without primer
Haemin, in DMSO (1 mM stock) 1 μL 1 μL
ddH₂O 98.5 μL 98.5 μL
TMB (50 mM stock in DMSO) 1.5 μL 1.5 μL
H₂O₂ 1.5 μL 1.5 μL
TOTAL 125 μL 125 μL

[P18]

See experiment E2.4

  1. Into each PCR tube, add:
(1) (2) (3) control (4)
10× G-quadruplex buffer 12.5 μL 12.5 μL 12.5 μL 12.5 μL
G-quadruplex sample (from IDT G-quad 8 µM stock) 18.75 μL 12.5 μL 6.25 μL 0.625 μL
Haemin, in DMSO (1 mM stock) 1 μL 1 μL 1 μL 1 μL
ddH₂O 89.75 μL 96 μL 102.25 μL 107.875 μL
TMB (50 mM stock in DMSO) 1.5 μL 1.5 μL 1.5 μL 1.5 μL
H₂O₂ 1.5 μL 1.5 μL 1.5 μL 1.5 μL
TOTAL 125 μL 125 μL 125 μL 125 μL

[P19]

See experiment E2.5

  1. Into a PCR tube, add:
Sample 1
RCA linear template (v3) (100 µM stock) 2 μL
Linear scaffold DNA (v3) (100 µM stock) 2 μL
ddH₂O 13 μL
TOTAL 17 μL
  1. Incubate the reaction mixture at 95°C for 5 minutes; set the thermocycler to gradually cool down to 25°C.
  2. Into the same PCR tube, add:
Sample 1
Reaction mixture from step 1 17 μL
10× T4 Ligase Buffer 2 μL
T4 Ligase 1 μL
TOTAL 20 μL
  1. Incubate at 22°C for 15 minutes.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 20 μL ddH₂O preheated at 65°C.

[P20]

See experiment E2.5

  1. Into the PCR tubes, add:
(1) RCA (v3) cut with Nt.BsmAI (2) RCA (v3) uncut
ddH₂O 11.5 μL 12 μL
rCutSmart Buffer 2 μL 2 μL
dNTP mix (2.5 mM each, stock) 2 μL 2 μL
RCA template (v3) (1 ng/μL stock) 1 μL 1 μL
RCA primer (10 µM stock) 2 μL 2 μL
TOTAL 18.5 μL 19 μL
  1. Incubate at 95°C for 5 minutes, and then return to ice.
  2. Into the same PCR tubes, add:
(1) RCA (v3) cut with Nt.BsmAI (2) RCA (v3) uncut
Product from previous steps 18.5 μL 19 μL
Φ29 DNA polymerase 1 μL 1 μL
Nt.BsmAI 0.5 μL -
TOTAL 20 μL 20 μL
  1. Incubate at 37°C for 2 hours.
  2. Perform heat inactivation at 65°C for 20 minutes.
  3. Run 10 μL of each sample to gel electrophoresis using 0.8% Agarose/TAE gel with GelGreen.

[P21]

See experiment E2.5

  1. Into each PCR tube, add:
(1) RCA(v3)/Nt.BsmAI-GQuad (2) RCA(v3)-GQuad (+) control
10× G-quadruplex buffer 12.5 μL 12.5 μL 12.5 μL
G-quadruplex sample 10 μL 10 μL 6.25 μL
Haemin, in DMSO (1 mM stock) 1 μL 1 μL 1 μL
ddH₂O 98.5 μL 98.5 μL 102.25 μL
TMB (50 mM stock in DMSO) 1.5 μL 1.5 μL 1.5 μL
H₂O₂ 1.5 μL 1.5 μL 1.5 μL
TOTAL 125 μL 125 μL 125 μL

[P22]

See experiment E2.6
See Third-Generation RCA

  1. Into a PCR tube, add:
Sample 1
RCA linear template (v3) (100 µM stock) 2 μL
Linear scaffold DNA (v3) (100 µM stock) 2 μL
ddH₂O 13 μL
TOTAL 17 μL
  1. Incubate the reaction mixture at 95°C for 5 minutes; set the thermocycler to gradually cool down to 25°C.
  2. Into the same PCR tube, add:
Sample 1
Reaction mixture from step 1 17 μL
10× T4 Ligase Buffer 2 μL
T4 Ligase 1 μL
TOTAL 20 μL
  1. Incubate at 22°C for 15 minutes.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 18 μL ddH₂O preheated at 65°C.
  3. Into a new PCR tube, add:
Sample 1
ddH₂O 70.5 μL
Oligonucleotide purification product 18 μL
10× Exonuclease III buffer 10 μL
Exonuclease III (TaKaRa Bio) 0.5 μL
Exonuclease I (Thermo Fisher Scientific) 1 μL
TOTAL 100 μL
  1. Incubate at 37°C for 1 hour.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 20 μL ddH₂O preheated at 65°C.
  3. Measure the sample concentration and dilute to 1 ng/μL.

[P23]

See experiment E2.6
See Third-Generation RCA

  1. Into the PCR tubes, add:
(1) 1 ng (2) 2 ng (3) 4 ng (4) 8 ng (5) uncut (6) (-)
ddH₂O 11.5 μL 10.5 μL 8.5 μL 4.5 μL 12 μL 14 μL
rCutSmart Buffer 2 μL 2 μL 2 μL 2 μL 2 μL 2 μL
dNTP mix (10 mM each, stock) 2 μL 2 μL 2 μL 2 μL 2 μL 2 μL
RCA template (v3) (1 ng/μL stock) 1 μL 2 μL 4 μL 8 μL 1 μL 1 μL
RCA primer (10 µM stock) 2 μL 2 μL 2 μL 2 μL 2 μL -
TOTAL 18.5 μL 18.5 μL 18.5 μL 18.5 μL 19 μL 19 μL
  1. Incubate at 95°C for 5 minutes, and then return to ice.
  2. Into the same PCR tubes, add:
(1) 1 ng (2) 2 ng (3) 4 ng (4) 8 ng (5) uncut (6) (-)
Product from previous steps 18.5 μL 18.5 μL 18.5 μL 18.5 μL 19 μL 19 μL
Φ29 DNA polymerase 1 μL 1 μL 1 μL 1 μL 1 μL 1 μL
Nt.BsmAI 0.5 μL 0.5 μL 0.5 μL 0.5 μL - -
TOTAL 20 μL 20 μL 20 μL 20 μL 20 μL 20 μL
  1. Incubate at 37°C for 2 hours.
  2. Perform heat inactivation at 65°C for 20 minutes.
  3. Run 10 μL of each sample to gel electrophoresis using 0.8% Agarose/TAE gel with GelGreen.

[P24]

See experiment E2.6
See Signal Expression: G-Quadruplex DNAzyme Assay

  1. Into each PCR tube, add:
(1) 1 ng (2) 2 ng (3) 4 ng (4) 8 ng (5) uncut (6) (-) (7) (+) control
10× G-quadruplex buffer 12.5 μL 12.5 μL 12.5 μL 12.5 μL 12.5 μL 12.5 μL 12.5 μL
G-quadruplex sample 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 6.25 μL
Haemin, in DMSO (1 mM stock) 1 μL 1 μL 1 μL 1 μL 1 μL 1 μL 1 μL
ddH₂O 98.5 μL 98.5 μL 98.5 μL 98.5 μL 98.5 μL 98.5 μL 102.25 μL
TMB (50 mM stock in DMSO) 1.5 μL 1.5 μL 1.5 μL 1.5 μL 1.5 μL 1.5 μL 1.5 μL
H₂O₂ 1.5 μL 1.5 μL 1.5 μL 1.5 μL 1.5 μL 1.5 μL 1.5 μL
TOTAL 125 μL 125 μL 125 μL 125 μL 125 μL 125 μL 125 μL

[P25]

See Cell Lysis: SDS Treatment

  1. Into each PCR tube, add:
SDS-Treated Control (Untreated)
Cell culture 50 μL 50 μL
1× SDS 50 μL -
1× Neutralisation buffer 75 μL -
ddH₂O - 125 μL
TOTAL 175 μL 175 μL
  1. Mix all tubes by flicking.
  2. Spin down in a microcentrifuge for 2 minutes.
  3. Mix 50 μL of each sample of cell culture with 50 μL of glycerol.
  4. Add 3 μL of each sample from step (4) on a microscope slide.
  5. Secure the cover slip on top of the microscope slide and observe the cells under the microscope.

[P26]

See Target Gene Extraction: Nt.BsmAI/HhaI-LSDA

  1. Into each PCR tube, add:
Nt.BsmAI & HhaI HhaI
ddH₂O 0.32 μL 0.66 μL
rCutSmart Buffer 2 μL 2 μL
Nt.BsmAI 0.34 μL -
HhaI 0.34 μL 0.34 μL
pSB1C3-KPC-2 (68.4 ng/μL stock) 14 μL 14 μL
TOTAL 17 μL 17 μL
  1. Incubate at 37°C for 45 minutes.
  2. Into the same PCR tubes, add:
Nt.BsmAI & HhaI HhaI
Sample from step (2) 17 μL 17 μL
Φ29 DNA polymerase 1 μL 1 μL
dNTP 2 μL 2 μL
TOTAL 20 μL 20 μL
  1. Incubate at 30°C for 15 minutes.
  2. Perform heat inactivation at 65°C for 20 minutes.
  3. Run 10 μL of each sample to gel electrophoresis using 5% Agarose/TAE gel with GelGreen.

[P27]

See RCA in rCutSmart

  1. Into a PCR tube, add:
Sample 1
RCA linear template (v1) (100 µM stock) 2 μL
Linear scaffold DNA (v1) (100 µM stock) 2 μL
ddH₂O 13 μL
TOTAL 17 μL
  1. Incubate the reaction mixture at 95°C for 5 minutes; set the thermocycler to gradually cool down to 25°C.
  2. Into the same PCR tube, add:
Sample 1
Reaction mixture from step 1 17 μL
10× T4 Ligase Buffer 2 μL
T4 Ligase 1 μL
TOTAL 20 μL
  1. Incubate at 22°C for 15 minutes.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 17 μL ddH₂O preheated at 65°C.
  3. Into a new PCR tube, add:
Sample 1
Oligonucleotide purification product 17 μL
10× Exonuclease I reaction buffer 2 μL
Exonuclease I 1 μL
TOTAL 20 μL
  1. Incubate at 37°C for 2 hours.
  2. Perform oligonucleotide purification protocol to the reaction mixture, and elute with 20 μL ddH₂O preheated at 65°C.
  3. Measure the sample concentration and dilute to 1 ng/μL.

[P28]

See RCA in rCutSmart

  1. Into the PCR tubes, add:
10× Reaction Buffer rCutSmart Buffer
ddH₂O 11.4 μL 13.4 μL
Buffer 2 μL 2 μL
dNTP mix (10 mM each, stock) 2 μL 2 μL
RCA template (v1) (1 ng/μL stock) 1 μL 1 μL
RCA primer (10 µM stock) 2 μL -
DTT (0.1 M stock) 0.6 μL 0.6 μL
TOTAL 19 μL 19 μL
  1. Incubate at 95°C for 5 minutes, and then return to ice.
  2. Add 1 μL of Φ29 DNA polymerase to each tube.
  3. Incubate at 30°C for 2 hours.
  4. Perform heat inactivation at 65°C for 10 minutes.
  5. Run 10 μL of each sample to gel electrophoresis using 0.8% Agarose/TAE gel with GelGreen.

Buffer Preparation Protocols

G-Quadruplex Buffer Preparation (10×) (in 1L)

Reagent Quantity
Tris 0.1 M (100 mL of 1M Tris)
Triton X-100 0.5% (5 mL of 1M Triton X-100)
DMSO 10% (100 mL of 1M DMSO)
KCl 1 M (500 mL of 2M KCl)
ddH₂O Variable
HCl Variable
  1. Mix the first 4 components as per the final concentrations listed in the table.
  2. Top up to 800 mL using ddH₂O.
  3. Adjust to pH 7.5 with HCl.
  4. Top up to 1000 mL using ddH₂O.

13PD1 DNAzyme Buffer (1×) (in 1L)

Reagent Quantity
ZnCl₂ 0.01 M (10 mL of 1 M ZnCl₂)
MnCl₂ 0.2 M (200 mL of 1 M MnCl₂)
MgCl₂ 0.1 M (100 mL of 1 M MgCl₂)
NaCl 0.375 M (75 mL of 5 M NaCl)
MOPS 0.1 M (100 mL of 1 M MOPS)
ddH₂O Variable
NaOH Variable
  1. Mix the first 5 components as per the final concentrations listed in the table.
  2. Top up to 800 mL using ddH₂O.
  3. Adjust to pH 7.5 with NaOH.