Biomod/2013/IIT-Madras/labnote

From OpenWetWare
Jump to navigationJump to search

Equipment Required

For Phase 1:

  • Nano-drop Spectrophotometer
  • Gel apparatus (for Native PAGE)
  • Falcons, chemicals for Buffer preparation

For Phase 2:

  • Spectrofluorometer
  • Heat Blocks
  • Water Bath (if possible at T = 25°C)
  • Fluorescence Microscopy
  • AFM (If possible)


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

  1. ddH2O was added to all nucleotides in order to make their concentration up to 100 µM.
  2. Stock S2, was prepared by diluting the above stock solutions by a factor of 100.
  3. All the stock solutions were labelled and kept in the Deep Freeze
  4. 3M KCl solution was prepared by adding 2.24g KCl salt to 10ml ddH2O
  5. potassium phosphate buffer at pH 8 was prepared
  6. Solution of 1ml, 1M K2HPO4 was prepared by adding 0.174g of the salt to 1ml of water
  7. Solution of 0.5ml, 1M KH2PO4 was prepared by adding 0.068g of the salt to 0.5ml of water
  8. 940µL of K2HPO4 and 60µL of KH2PO4 were mixed together to form the buffer 0.1M
  9. 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)

  1. All the S2 stock solutions were quantified using NanoDrop spectrophotometer.
  2. Reading for I2 stock was off the required 1μM concentration. So, 1uM I2 stock was prepared again in a 1.5 ml Eppendorf Tube
  3. Annealing mixtures were prepared - BI1I2, I1L1, BL2I2, L2I2, L2B by using the composition as given in the protocol in 0.5 ml Eppendorf Tubes
  4. The 5 samples were annealed in a heat block - Kept at 90C for 5 min
  5. This was followed by a drop of 5C every 15 minutes. This was done until the temperature reached 30C


4th September, 2013

Materials used: Gel loading buffer (in Eppendorf Tube)
2 Eppendorf tubes(2ml)
Conical Flask for gel running buffer preparation
1 empty 15ml Falcon tube needs to be autoclaved

  1. The percentage of PAGE to be run was decided to be 20%
  2. A solution of 5X TBE buffer was prepared to use for gel preparation in a 50ml Falcon Tube consisting of

1.63g Tris Base 0.825g Boric Acid 600 µL 0.5M EDTA solution

  1. A 2ml Eppendorf tube was used for EDTA preparation and another was used to aliquot EDTA stock.
  2. Gel loading buffer was taken from the lab in an Eppendorf tube
  3. Gel Running Buffer was prepared - 200ml 5X TBE buffer

10.8g Tris Base 5.5g Boric Acid 4ml 0.5M EDTA solution

  1. The pH of the buffer was checked using pH meter and found out to be equal to 8.46
  2. The buffer was stored in a conical flask


5th September, 2013

  1. A PAGE(20%) was run with the lanes - I1L1 anealed, I1, L1, BI2L2 annealed, BL2annealed, L2I2 annealed. It was stored in water.
  2. EtBr stock solution (10mg/ml) was prepared by weighing approximately 4.7 mg of EtBr
  3. The gel on staining showed no bands (100ml water + EtBr 10uL)
  4. Possibly, this was due to the improper placement of the dummy plate.
  5. Buffer solution was diluted using can water
  6. The buffer used was stored for future use

20% PAGE components 6.6ml of 29:1 acrylamide 0.07ml of APS 2ml of 5X TBE Buffer 2.3ul of TEMED Water to make upto 10ml


6th September 2013

Gel Running Buffer was prepared - 200ml 5X TBE buffer 10.8g Tris Base 5.5g Boric Acid 4ml 0.5M EDTA solution

Buffer pH was quantified

Another PAGE gel was cast . PAGE components 6.6ml of 29:1 acrylamide 0.07ml of APS 2ml of 5X TBE Buffer 2.3ul of TEMED Water to make upto 10ml

Lanes loaded - B2(2, 4, 8, 16, ul), I1, L1, I1L1 annealed. Run at 70V in cold room.

The last 3 bands were seen distinctly. The first ones were unclear after staining with 50ml 1X TBE + 10uL EtBr. Hence, the gel was stained again with 1uL EtBr This caused the whole gel to be stained with EtBr. Hence, further gels need to be run. 20th September, 2013

A gel was run to confirm formation of our final structure

A 10mL gel of 15% acrylamide was prepared 5ml of 29:1 acrylamide 0.07ml of APS 2ml of 5X TBE Buffer 10ul of TEMED Water to make upto 10ml

The lanes loaded were - I1, L1, I1L1 annealed, BI1I2 annealed, BL2 annealed, L2I2 annealed, BL2I2 annealed, BL2I2 annealed + I1L1 annealed

The tank was filled with pre chilled buffer. Samples were prepared and loaded in the lab. The gel was kept in the cold room for running.

The gel was run at 70V for about half an hour

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.

The power was switched off. The gel was imaged by placing in 50mL water + 10uL EtBr and shaking 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

1.67uL - KCl soln + 5uL of repsective nucleotide stock (S2) + Potassium Phosphate Buffer to make up to 50uL - Anneal in KG programme

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Biomod/2013/IIT-Madras/labnote&oldid=746959"