Reaction No. | [3×pen aRNA] (μM)* | Molar ratio 3×pen aRNA: Pen17zyme1/1B/1C: Pen17zyme2 | Additional components in the annealing buffer | [Mn2+] (mM)* | Reaction T (°C) | Time | Additional Cycles |
---|---|---|---|---|---|---|---|
1 | 0.25 | 1:0:1 | N/A | 0 | 23 | 10 min | 0 |
2 | 0.25 | 1:0:1 | N/A | 7.5 | 23 | 10 min | 0 |
3 | 0.25 | 1:0:1 | N/A | 15 | 23 | 10 min | 0 |
4 | 0.25 | 1:0:1 | N/A | 45 | 23 | 10 min | 0 |
5 | 0.25 | 1:0:1 | N/A | 15 | 23 | 100 min | 0 |
6 | 0.25 | 1:1(Pen17zyme1):0 | N/A | 15 | 23 | 100 min | 0 |
7 | 0.25 | 1:1(Pen17zyme1B):0 | N/A | 15 | 23 | 100 min | 0 |
8 | 0.25 | 1:1(Pen17zyme1C):0 | N/A | 15 | 23 | 100 min | 0 |
9 | 0.25 | 1:5(Pen17zyme1B):0 | N/A | 15 | 23 | 100 min | 0 |
10 | 0.25 | 1:1(Pen17zyme1B):0 | N/A | 15 | 23 | 17 h | 0 |
11 | 0.25 | 1:1(Pen17zyme1B):0 | N/A | 15 | 40 | 100 min | 0 |
12 | 0.25 | 1:1(Pen17zyme1B):0 | 10%EtOH | 15 | 23 | 100 min | 0 |
13 | 0.25 | 1:5(Pen17zyme1B):0 | 0.3 M KCl, 1.2 M NaCl | 15 | 23 | 17 h | 0 |
14 | 0.25 | 1:5(Pen17zyme1B):0 | 20% PEG 8000 | 15 | 23 | 17 h | 0 |
15 | 0.25 | 1:5(Pen17zyme1B):0 | 500 μM spermine | 15 | 23 | 17 h | 0 |
16 | 0.25 | 1:5(Pen17zyme1B):0 | N/A | 15 | 23 | 17 h | 50 |
17 | 0.25 | 1:5(Pen17zyme1B):0 | 2.2 M potassium acetate | 15 | 23 | 17 h | 0 |
18 | 0.25 | 1:5(Pen17zyme1B):0 | 3 M LiCl | 15 | 23 | 17 h | 0 |
19 | 0.80 | 1:1(Pen17zyme1B):1 | 500 μM spermine | 15 | 23 | 17 h | 0 |
20 | 0.80 | 1:10(Pen17zyme1B):1 | 500 μM spermine | 15 | 23 | 17 h | 0 |
21 | 0.80 | 1:20(Pen17zyme1B):2 | 500 μM spermine | 15 | 23 | 17 h | 0 |
22 | 0.80 | 1:10(Pen17zyme1B):10 | 500 μM spermine | 15 | 23 | 17 h | 0 |
23 | 0.80 | 1:10(Pen17zyme1B):10 | 500 μM spermine | 15 | 23 | 17 h | 6 |
24 | 0.80 | 1:10(Pen17zyme1B):10 | 500 μM spermine | 15 | 23 | 40 h | 0 |
25 | 0.80 | 1:10(Pen17zyme1B):10 | 500 μM spermine | 15 | 23 | 72 h | 0 |
26 | 0.80 | 1:10(bioPen17zyme1B):10(biopen17zyme2) | 500 μM spermine | 15 | 23 | 72 h | 0 |