PCR amplify (30 cycles, 60C) target DNA using one fluorophore-labelled primer (e.g., Cy5):
Component
Calculation (uL)
Common vol. (uL)
Milli-Q water
to V
12.4
5X Phusion™ Plus Buffer
V / 5
4
dNTPs (10 mM)
V / 50
.4
Forward primer: 1.52 - NP1 (10 uM)
V / 20
1
Reverse primer: 5.19 - NP2 labelled with ATTO647N (10 uM)
V / 20
1
Template: 1.59 - Perfect target D (1 uM)
1
1
Phusion™ Plus DNA Polymerase
V/100
.2
Total
V
20
Purify PCR product and measure DNA concentration using Qubit.
Nanodrop is okay if you don't have access to a Qubit.
Dilute DNA to 20 nM (will be 10 nM in the final samples) with the 10X NucleaSeq buffer for final volume of 100 uL.
Form the RNP complex: Use 1:3 enzyme-to-gRNA ratio and incubate for 30 min at room temperature.
Component
Vol. (uL)
Input conc. (uM)
Final conc. (uM)
Milli-Q
to V
10X NucleaSeq buffer
V / 10
10X
1X
R7 gRNA (C12a U)
3 x V / [gRNA]
[gRNA]
3
AsCas12a-TwinStrep
V / [Cas]
[Cas]
1
Total
V
Serially dilute RNP:
The first sample (ID 1) is just a NucleaSeq buffer. We'll add target DNA to it in Step 6 and this will be a control condition where no DNA cleavage will happen.
The second sample (DI 2) uses 10 uL (column "Vol. from previous dilution (uL)") of the RNP complex produced in Step 4.
All subsequent samples use the indicated volume from the previous RNP dilution, e.g., sample 3 uses 4.5 uL of sample 2.
Best practice is to do serial dilutions in a row, starting with the greatest concentration and diluting by similar amounts.
ID
Dilution factor
Vol. from previous dilution (uL)
1X NucleaseSeq buffer (uL)
Input conc. (nM)
Final conc. (nM)
Conc. in exp. (nM)
DNA:RNP conc. ratio (folds)
1 (buffer only)
-
10
-
2
1
10
5
1000
666.67
333.33
.03
3
3.3333
4.50
10.50
666.67
200.00
100.00
0.10
4
10
5.00
10.00
200.00
66.67
33.33
0.3
5
33.333
4.50
10.50
66.67
20.00
10.00
1.00
6
100
5.00
10.00
20.00
6.67
3.33
3.00
7
333.33
4.50
10.50
6.67
2.00
1.00
10.00
8
1000
5.00
10.00
2.00
0.67
0.33
30.00
Total volume
15
DNA conc. in exp.
10
Perform cleavage:
Combine 10 uL of a sample with 10 uL of target DNA and incubate at 37C for 30 min.
Add 5 uL of Stop solution to each sample.
Incubate at 37C for 30 min.
Visualize on a gel:
12% native PAGE:
Milli-Q: 6 mL
10X TBE: 1 mL
40% acrylamide/bis (29:1): 3 mL
10% APS: 80 uL
TEMED: 10 uL
Sample: 5 uL.
Loading dye: 1 uL 6X OrangeG.
Pre-run: 10 min at 100V.
Run: 35 min at 200V – until all OrangeG is out of the gel.
Quantify the volume of each band on the gel using Image Lab or similar software and compute the fraction of cut DNA.
Plot the amount of cleavage by plotting RNP:DNA concentration (in folds) vs the fraction of cut DNA.
Enzyme activity is the lowest amount of RNP that completely cleaves target DNA. In practice, it is possible that the activity may plateau before full cleavage, in which case we treat the onset of plateau as enzyme's activity.
If the activity is above 1, this means that the enzyme exhibits a multi-turnover activity.