Biomod/2013/IIT-Madras/labnote

Equipment Required

• Spectrophotometer
• Nano-drop
• Spectrofluorometer
• Heat Blocks
• Water Bath (if possible at T = 25°C)
• Falcons, chemicals for Buffer preparation
• Gel apparatus (for Native PAGE)
• Fluorescence Microscopy
• AFM (If possible)

2nd September, 2013

ddWater 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 ddWater

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

940uL of K2HPO4 and 60uL 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

Things Taken

3 15mL Falcon Tubes - Used for KCl stock, phosphate buffer 2 50mL Falcon Tubes - Used for ddH2O 7 1.5 mL Eppendorf Tubes for S2 preparation 3 2mL Eppendorf Tubes for Potassium phosphate buffer preparation

3rd September, 3013

All the S2 stock solutions were quantified using NanoDrop

Reading for I2 stock was off the required 1uM concentration

Hence, 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 Materials Taken - 1 1.5ml Eppendorf 5 2 ml Eppendorf Tubes 5 0.5ml Eppendorf Tubes

4th September, 2013

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 consisting of 1.63g Tris Base 0.825g Boric Acid 600 uL 0.5M EDTA solution

A 2ml Eppendorf Tube was used for EDTA preparation. Another was used for aliquoting EDTA stock.

Gel loading buffer was taken from the lab in an Eppendorf Tube

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

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

Materials Taken Gel loading buffer (in Eppendorf Tube) 2 2ml Eppendorf Tubes Conical Flask for gel running buffer preparation

1 empty 15ml Falcon tube needs to be autoclaved

5th September, 2013

A PAGE was run (20%), with the lanes - I1L1 anealed, I1, L1, BI2L2 annealed, BL2annealed, L2I2 annealed. It was stored in water.

EtBr stock solution (10mg/ml) was prepared by weighing approximately 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 using can water

The buffer used was stored for further usage

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