Template:Heidelberg/notebook/pc/week36
week number 36
▼2015-08-31 Labeling of RNA with alkyne-NTP
The labeling RNA was modified with alkyne NTPs by Terminale Dinucleotidyl Transferase (TdT) and as a second approach, by Poly A Polymerase (yeast), affimetrix.
The modified NTPs are from Jena Bioscience:
- 5-Ethynyl-dUTP (5-EdUTP)
- C8-Alkyne-dUTP
- C8-Alkyne-dCTP
|
cStock |
cFinal |
V[µL] |
|
|
TdT buffer |
10 x |
1 x |
2.5 |
|
Labeling RNA |
84,97µM |
15 µM |
4.41 |
|
Alkyne-NTP |
10 mM |
400 µM |
1 |
|
TdT |
20,000 u/mL |
80 u/mL |
1 |
|
CoCl2 |
2.5 mM |
0.25 mM |
2.5 |
|
H2O |
Add 25 µL |
13.59 |
|
|
Final |
25 |
|
cStock |
cFinal |
V[µL] |
|
|
PAP (yeast) |
600 u/µL |
24 u/µL |
1 |
|
PAP buffer |
5 x |
1 x |
5 |
|
5-EdUTP/ C8-Alkyne-dUTP/C8-Alkyne-dCTP |
10 mM |
400 µM |
1 |
|
Labeling RNA |
84.97µM |
15µM |
4.41 |
|
H2O |
Add 25 µL |
13.59 |
|
|
Final |
25 |
- All compounds were mixed as indicated in the tables.
- Reactions were incubated for 2 hours at 37 °C
- Samples were stored at -20 °C oN
▼2015-09-01 Biotin modification of substrate RNA
|
For 25µL |
Cstock |
Cfinal |
V [µL] |
|
TdT- Buffer |
10x |
1x |
2.5 |
|
RNA |
71.804µM |
10µM |
3.48 |
|
Biotin - NTP |
1mM |
200µM |
5 |
|
TdT |
600U/µL |
24U/µL |
1 |
|
CoCl2 |
2.5mM |
0.5mM |
5 |
|
MQ H2O |
|
|
8.02 |
All samples were mixed together and incubated for 2 hours at 37 °C.
▼2015-09-01 Click reaction (modified)
- new Alexa 488 with DMSO
|
For 50µL |
Cstock |
Cfinal |
V[µL] |
|
Phosphate Buffer - pH 7, 0.1M |
100mM |
50mM |
25 |
|
Alexa 488 azide |
10µM |
400nM |
2 |
|
RNA |
1µM |
200nM |
10 |
|
CuSO4 |
50mM |
1mM |
1 |
|
THPTA |
50mM |
5mM |
5 |
|
NaAsc |
100mM |
1mM |
0.5 |
|
H2O |
|
|
6.5 |
All samples were mixed together and then incubated for 2hours at 37 °C.
▼2015-09-02 In vitro transcription of Labeling RNA AU + substrate HRP
|
cStock |
cFinal |
V[µL] |
|
|
Transcription buffer |
10 x |
1 x |
20 |
|
ATP |
100 mM |
4 mM |
8 |
|
GTP |
100 mM |
4 mM |
8 |
|
CTP |
100 mM |
4 mM |
8 |
|
UTP |
100 mM |
4 mM |
8 |
|
DTT |
1 M |
10 mM |
2 |
|
DMSO |
5 % |
10 |
|
|
FS_53/54 (Label AU) |
|
|
20 |
|
T7 RNA Polymerase |
2 mg/mL |
0,1 mg/mL |
5 |
|
H2O |
ad 100 µL |
106 |
|
|
Final |
400 |
|
cStock |
cFinal |
V[µL] |
|
|
Transcription buffer |
10 x |
1 x |
20 |
|
ATP |
100 mM |
4 mM |
8 |
|
GTP |
100 mM |
4 mM |
8 |
|
CTP |
100 mM |
4 mM |
8 |
|
UTP |
100 mM |
4 mM |
8 |
|
DTT |
1 M |
10 mM |
2 |
|
DMSO |
5 % |
10 |
|
|
FS_50/51 (Substrate HRP) |
|
|
20 |
|
T7 RNA Polymerase |
2 mg/mL |
0,1 mg/mL |
5 |
|
H2O |
ad 100 µL |
106 |
|
|
Final |
200 |
All compounds were mixed together and incubated for 3 hours at 37 °C. Afterwards, 2µL DNAseI was added for 30min at 37 °C.