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Revision as of 09:56, 15 September 2015
week number 38
▼2015-09-14 Test bitte löschen
10 x <i>in vitro</i> transcription buffer
50 mM Tris ph 7.5, 100 mM NaCl, 20 mM MgCl<sub>2</sub>, 0,01 % SDS
week number 37
▼2015-09-10 DNAzyme Activity: DNAzyme with ATP aptamer and calculated Kanamycin aptamer
Samples:
- 10-23 DNAzyme: xxfs032xx
- 7-18 DNAzyme: xxfs033xx
- 10-23 DNAzyme with ATP aptamer with linker: xxfs019xx
- 7-18 DNAzyme with ATP aptamer with linker: xxfs027xx
- 10-23 DNAzyme with ATP aptamer A: xxfs017xx, B: xxfs018xx
- 7-18 DNAzyme with ATP aptamer A: xxfs025, B: xxfs026xx
- 10-23 DNAzyme with calculated Kan aptamer: xxfs034xx
- 7-18 DNAzyme with calculated Kan aptamer candidate I: xxfs035xx
- 7-18 DNAzyme with calculated Kan aptamer candidate II: xxfs036xx
- 7-18 DNAzyme with calculated Kan aptamer candidate III: xxfs037xx
Stock solutions and conditions:
|
cStock |
cFinal |
|
|
Tris HCl ph 7.5 |
1 M |
50 mM |
|
DNAzyme (A) |
10 µM |
500 nM |
|
DNAzyme B |
10 µM |
500 nM |
|
Substrate |
1 µM |
200 nM |
|
NaCl |
1 M |
100 mM |
|
MgCl2 |
1 M |
20 mM |
|
SDS |
20 % |
0,01 % |
|
Adenosine in H2O:DMSO 1:2 |
33 mM |
5 mM |
|
H2O |
|
ad 25 µL |
Pipetting scheme:
|
# |
|
|
Tris HCl ph 7.5 |
DNAzyme A |
DNAzyme B |
Substrate |
NaCl |
MgCl2 |
SDS |
Adenosine |
H2O |
Final |
|
1 |
FS032 |
10-23D |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
3,75 |
9,50 |
25,00 |
|
2 |
FS033 |
7-18D |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
3,75 |
9,50 |
25,00 |
|
3 |
FS019 |
10-23DmLink |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
3,75 |
9,50 |
25,00 |
|
4 |
FS027 |
7-18DmLink |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
3,75 |
9,50 |
25,00 |
|
5 |
FS017+18 |
10-23D_A+B |
1,25 |
1,25 |
6,25 |
5,00 |
2,50 |
0,50 |
1,25 |
3,75 |
3,25 |
25,00 |
|
6 |
FS025+26 |
7-18D_A+B |
1,25 |
1,25 |
6,25 |
5,00 |
2,50 |
0,50 |
1,25 |
3,75 |
3,25 |
25,00 |
|
7 |
FS032 |
10-23D |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
0,00 |
13,25 |
25,00 |
|
8 |
FS033 |
7-18D |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
0,00 |
13,25 |
25,00 |
|
9 |
FS019 |
10-23DmLink |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
0,00 |
13,25 |
25,00 |
|
10 |
FS027 |
7-18DmLink |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
0,00 |
13,25 |
25,00 |
|
11 |
FS017+18 |
10-23D_A+B |
1,25 |
1,25 |
6,25 |
5,00 |
2,50 |
0,50 |
1,25 |
0,00 |
7,00 |
25,00 |
|
12 |
FS025+26 |
7-18D_A+B |
1,25 |
1,25 |
6,25 |
5,00 |
2,50 |
0,50 |
1,25 |
0,00 |
7,00 |
25,00 |
|
13 |
FS032 |
-10-23D |
1,25 |
1,25 |
0 |
0 |
2,5 |
0,5 |
1,25 |
0 |
18,25 |
25,00 |
|
14 |
FS033 |
-7-18D |
1,25 |
1,25 |
0 |
0 |
2,5 |
0,5 |
1,25 |
0 |
18,25 |
25,00 |
|
15 |
FS019 |
-10-23DmLink |
1,25 |
1,25 |
0 |
0 |
2,5 |
0,5 |
1,25 |
0 |
18,25 |
25,00 |
|
16 |
FS027 |
-7-18DmLink |
1,25 |
1,25 |
0 |
0 |
2,5 |
0,5 |
1,25 |
0 |
18,25 |
25,00 |
|
17 |
FS017+18 |
-10-23D_A+B |
1,25 |
1,25 |
6,25 |
0 |
2,5 |
0,5 |
1,25 |
0 |
12 |
25,00 |
|
18 |
FS025+26 |
-7-18D_A+B |
1,25 |
1,25 |
6,25 |
0 |
2,5 |
0,5 |
1,25 |
0 |
12 |
25,00 |
|
19 |
Adenosine |
Substrate only |
1,25 |
0 |
0 |
5 |
2,5 |
0,5 |
1,25 |
3,75 |
10,75 |
25,00 |
|
# |
Tris HCl ph 7.5 |
DNAzyme A |
DNAzyme B |
Substrate |
NaCl |
MgCl2 |
SDS |
Kan |
H2O |
Final |
||
|
20 |
FS032 |
10-23D |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
1,25 |
12 |
25 |
|
21 |
FS033 |
7-18D |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
1,25 |
12 |
25 |
|
22 |
FS034 |
Kan |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
1,25 |
12 |
25 |
|
23 |
FS035 |
Kan I |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
1,25 |
12 |
25 |
|
24 |
FS036 |
Kan II |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
1,25 |
12 |
25 |
|
25 |
FS037 |
Kan III |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
1,25 |
12 |
25 |
|
26 |
FS032 |
10-23D |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
0 |
13,25 |
25 |
|
27 |
FS033 |
7-18D |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
0 |
13,25 |
25 |
|
28 |
FS034 |
Kan |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
0 |
13,25 |
25 |
|
29 |
FS035 |
Kan I |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
0 |
13,25 |
25 |
|
30 |
FS036 |
Kan II |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
0 |
13,25 |
25 |
|
31 |
FS037 |
Kan III |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
0 |
13,25 |
25 |
|
32 |
Kan |
Substrate only |
1,25 |
0 |
0 |
5 |
2,5 |
0,5 |
1,25 |
1,25 |
13,25 |
25,00 |
|
33 |
Substrate only |
1,25 |
0 |
0 |
5 |
2,5 |
0,5 |
1,25 |
0 |
14,5 |
25,00 |
Results and Outlook:
Positive controls worked, Adenosine dependency could be detected for one candidate.
▼2015-09-10 Click reaction of Label-, Label AU RNA [1, 2, 3]
|
For 51.5µL |
Cstock |
Cfinal |
V[µL] |
|
Phosphate Buffer - pH 7, 0.1M |
100mM |
50mM |
25 |
|
Alexa 488 azide |
10µM |
400nM |
2 |
|
RNA |
1µM |
200nM |
10 |
|
CuSO4 |
20mM |
1mM |
2.5 |
|
THPTA |
50mM |
5mM |
5 |
|
NaAsc |
100mM |
1mM |
0.5 |
|
H2O |
|
|
6.5 |
- Alexa 488 azide was solved in DMSO
- Incubation at 37 °C for 12-14 hours (overnight)