Biomod/2013/IIT-Madras/labnote: Difference between revisions
T P Sanjan (talk | contribs) |
T P Sanjan (talk | contribs) |
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* Spectrofluorometer | * Spectrofluorometer | ||
* Heat Blocks | * Heat Blocks | ||
* Water | * Water bath (at 25°C) | ||
* Fluorescence Microscopy | * Fluorescence Microscopy | ||
* [http://en.wikipedia.org/wiki/Atomic_force_microscopy AFM] ( | * [http://en.wikipedia.org/wiki/Atomic_force_microscopy AFM] (if slot available) | ||
Line 55: | Line 55: | ||
Materials used:<br /> | Materials used:<br /> | ||
2 Eppendorf tubes(2ml)<br /> | 2 Eppendorf tubes(2ml)<br /> | ||
Conical flask for gel running buffer | Conical flask(for preparation of gel running buffer)<br /> | ||
1 autoclaved Falcon tube(15ml)<br /> | 1 autoclaved Falcon tube(15ml)<br /> | ||
Line 63: | Line 63: | ||
Reagent preparation:<br /> | Reagent preparation:<br /> | ||
5X TAE buffer: | 5X TAE buffer:<br /> | ||
1.63g Tris Base<br /> | 1.63g Tris Base<br /> | ||
0.825g Boric Acid<br /> | 0.825g Boric Acid<br /> | ||
600µL of 0.5M EDTA solution<br /> | 600µL of 0.5M EDTA solution<br /> | ||
5X TBE buffer: | 5X TBE buffer:<br /> | ||
10.8g Tris Base<br /> | 10.8g Tris Base<br /> | ||
5.5g Boric Acid<br /> | 5.5g Boric Acid<br /> |
Revision as of 12:45, 26 October 2013
Equipment Required
For Phase 1:
- Nano-drop Spectrophotometer
- Gel apparatus (for Native PAGE)
- Falcon tubes
For Phase 2:
- Spectrofluorometer
- Heat Blocks
- Water bath (at 25°C)
- Fluorescence Microscopy
- AFM (if slot available)
2nd September, 2013
Materials used:
3 Falcon tubes(15 mL) - Used for preparing KCl stock & phosphate buffer
2 Falcon tubes(50 mL) - Used for ddH2O
7 Eppendorf tubes(1.5 mL) - Used for S2 stock preparation
3 Eppendorf tubes(2 mL) - Used for Potassium Phosphate buffer preparation
- ddH2O was added to all nucleotides in order to make their concentration up to 100 µM.
- Stock S2, was prepared by diluting the above stock solutions by a factor of 100.
- All the stock solutions were labelled and kept in the Deep Freeze
- 3M KCl solution was prepared by adding 2.24g KCl salt to 10ml ddH2O
- potassium phosphate buffer at pH 8 was prepared
- Solution of 1ml, 1M K2HPO4 was prepared by adding 0.174g of the salt to 1ml of water
- Solution of 0.5ml, 1M KH2PO4 was prepared by adding 0.068g of the salt to 0.5ml of water
- 940µL of K2HPO4 and 60µL of KH2PO4 were mixed together to form the buffer 0.1M
- It was diluted 5 times, by adding 4ml of ddH2O to prepare a 20mM solution
3rd September, 2013
Materials used:
1 Eppendorf tube(1.5ml)
5 Eppendorf tubes (2 ml)
5 Eppendorf Tubes (0.5ml)
- All the S2 stock solutions were quantified using NanoDrop spectrophotometer.
- Reading for I2 stock was off the required 1μM concentration. So, 1uM I2 stock was prepared again in a 1.5 ml Eppendorf Tube
- Annealing mixtures were prepared - BI1I2, I1L1, BL2I2, L2I2, L2B by using the composition as given in the protocol in 0.5 ml Eppendorf Tubes
- The 5 samples were annealed in a heat block - Kept at 90C for 5 min
- This was followed by a drop of 5C every 15 minutes. This was done until the temperature reached 30C
4th September, 2013
Materials used:
2 Eppendorf tubes(2ml)
Conical flask(for preparation of gel running buffer)
1 autoclaved Falcon tube(15ml)
Reagent preparation:
5X TAE buffer:
1.63g Tris Base
0.825g Boric Acid
600µL of 0.5M EDTA solution
5X TBE buffer:
10.8g Tris Base
5.5g Boric Acid
4ml of 0.5M EDTA solution
- The percentage of PAGE to be run was decided to be 20%.
- A solution of 5X TBE buffer was prepared to use for gel preparation in a 50ml Falcon tube.
- A 2ml Eppendorf tube was used for EDTA preparation and another was used to aliquot EDTA stock.
- Gel loading buffer was taken from the lab in an Eppendorf tube.
- Gel Running Buffer was prepared - 200ml 5X TBE buffer.
- The pH of the buffer was checked using pH meter and found out to be equal to 8.46
- The buffer was stored in a conical flask
5th September, 2013
Reagent preparation:
20% PAGE components: 6.6ml of 29:1 acrylamide 0.07ml of APS 2ml of 5X TBE Buffer 2.3μl of TEMED Appropriate amount of water was added to make the final volume upto 10ml.
- A PAGE(20% acrylamide) was run by loading the following samples in different lanes - I1L1 annealed, I1, L1, BI2L2 annealed, BL2 annealed, L2I2 annealed.
- EtBr stock solution (10mg/ml) was prepared by weighing 4.7 mg of EtBr.
- The gel on staining showed no bands (100ml water + EtBr 10uL). Possibly, this was due to the improper placement of the dummy plate.
- Buffer solution was diluted and stored for future use.
6th September, 2013
Reagent preparation:
Gel Running Buffer/5X TBE buffer: 10.8g Tris Base 5.5g Boric Acid 4ml 0.5M EDTA solution Appropriate amount of water was added to make the final volume upto 200ml.
20% PAGE components: 6.6ml of 29:1 acrylamide 0.07ml of APS 2ml of 5X TBE Buffer 2.3ul of TEMED Appropriate amount of water was added to make the final volume upto 10ml.
- Buffer pH was quantified
- Another PAGE gel was cast.
- Samples loaded in different lanes - B2(2,4,8,16 μl), I1, L1, I1L1 annealed.
- The gel electrophoresis was carried out at 70V in cold room.
This time atleast the last 3 lanes showed bands that were distinctly visible !! The first few lanes were unclear after staining with 50ml of 1X TBE + 10μL EtBr solution. Hence, the gel was stained with 1μL EtBr solution again.
20th September, 2013
Reagent preparation:
15% PAGE components: 5ml of 29:1 acrylamide 0.07ml of APS 2ml of 5X TBE Buffer 10μl of TEMED Appropriate amount of water was added to make the final volume upto 10ml.
- A gel was run to confirm formation of our final structure.
- This time the lanes were loaded with following samples:
I1, L1, I1L1 annealed, BI1I2 annealed, BL2 annealed, L2I2 annealed, BL2I2 annealed, mixture of BL2I2 annealed & I1L1 annealed.
- The electrophoresis tank was filled with pre-chilled buffer.
- Later,the gel was run at 70V for about half an hour in the cold room.
- This caused the lanes in the centre to move faster compared to the lanes on the side of the gel, possibly due to heat generation.
- Then, the voltage was lowered to 60V for half an hour.
- Finally, it was kept at 50V for one more hour.
- After switching the power off, the gel was stained by placing in a solution of 50mL water & 10μL EtBr and kept in rocker for 15min.
Things to be done
Anneal a fresh set of samples
Prepare a 12% gel next time
Keep the running voltage low - Check Sambrook for the exact value (6V per cm of the electrode ??)
Run a 10bp ladder along with the samples - Will enable easy comparison with controls. Will tell us if gel running voltage or the sample preparation is wrong.
22nd September, 2013
- The following samples were annealed - I1L1, BI1I2, BL2I2, BL2, BI2 for which 5uL of repsective nucleotide stock (S2) was added to PCR tubes containing 1.67μl of KCl solution & Potassium Phosphate buffer(appropriate amount was added to make final volume upto 50μl).