Template:Heidelberg/notebook/ivt/week31
week number 31
▼2015-07-27 4. Elution and Ethanol Precipitation of Spinach2 RNA
Aim: Purification of the Spinach2 for further experiments
Procedure:
- RNA was eluted from the gel in 0.3 M sodium acetate pH 5.5
- 2.5x V of absolute ethanol was added
- RNA was incubated at -80 °C over night
- Precipitate the RNA at 16,000 g, 4 °C, 30 min
- Wash the RNA pellet in 70 % absolute ethanol and precipitate at 16,000 g (repeat three times)
- Resolve RNA in 100 µL millipore water
Result: The pellet was resuspended in 100 µL millipore water which will be the stock solution for further experiments.
▼2015-07-27 5. Analysis of the fluorescence of the Aptamer “Spinach2” in presence of the DFHBI dye
Aim: Showing the functionality of the Spinach2 in presence of DFHBI in 384-well plate format
Procedure:
- Measure the concentration of the RNA using a Nanodrop:
|
|
Concentration [µM] |
|
Spinach2 |
29.8 |
- Renaturing of RNA:
- Renature a 2 µM RNA stock with 1x Renaturing Buffer at 95 °C for 3 min
- Cool down to room temperature
- Spinach Assay:
|
|
cStock |
cfinal |
Volume [µL] |
|
HEPES KOH pH 7.5 |
1 M |
40 mM |
8 |
|
KCl |
2 M |
125 mM |
12.5 |
|
MgCl2 |
1 M |
3 mM |
0.3 |
|
DFHBI |
20 mM |
100 µM |
0.5 |
|
Ligand |
0.01 M |
100 µM |
1 |
|
RNA |
1 µM |
0.2 µM |
20 |
|
Millipore Water |
|
|
57.7 |
- Following conditions were tested:
|
|
Blank w/o DFHBI |
Blank w/o RNA |
Spinach2 |
|
HEPES KOH pH 7.5 |
8 µL |
8 µL |
8 µL |
|
KCl |
12.5 µL |
12.5 µL |
12.5 µL |
|
MgCl2 |
0.3 µL |
0.3 µL |
0.3 µL |
|
DFHBI |
|
0.5 µL |
0.5 µL |
|
RNA |
20 µL |
|
20 µL |
|
Millipore Water |
58.7 µL |
78.7 µL |
58.7 µL |
Conditions for the Tecan Safire 2:
- Excitation: 460 nm
- Emission: 500 nm
- Excitation Bandwidth: 10 nm
- Emission Bandwidth: 10 nm
- Gain: Manual
- Gain: 155
- Flash Mode: High Sensitivity
- Integration Time: 40 µs
- Lag Time: 0 µs
- Settle Time: 0 ms
- Reading Mode: Bottom
- Kinetic Cycle
- Number of Cycles: 50
- Interval: every 30 sec
- Total Time: 25 min
Results: The assay was performed with two controls. One sample did not contain DFHBI, the other one did not have the Spinach2 RNA. The blank with no DFHBI shows a low background, whereas the control with DFHBI shows a big background (RFU= 841). However the emission of the Spinach2 containing DFHBI shows higher values (RFU= 3586) than the controls, concluding the functionality of the Spinach2 construct.