Biomod/2013/IIT-Madras/labnote

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 October, 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. Call this stock S1.
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 October, 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 October, 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

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.
3. Gel loading buffer was taken from the lab in an Eppendorf tube.
4. Gel Running Buffer was prepared - 200ml 5X TBE buffer.
5. The pH of the buffer was checked using pH meter and found out to be equal to 8.46
6. The buffer was stored in a conical flask

5th October, 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.

1. 5X Running Buffer was diluted using can water
2. A PAGE(20% acrylamide) was run by loading the following samples in different lanes - I1L1 annealed, I1 (S2 stock), L1 (S2 stock), BI2L2 annealed, BL2 annealed, L2I2 annealed. This was done in order to detect formation of the structures I1L1 and BI2L2.
3. EtBr stock solution (10mg/ml) was prepared by weighing 4.7 mg of EtBr.
4. The gel on staining showed no bands (100ml water + EtBr 10uL). Possibly, this was due to the improper placement of the dummy plate during PAGE or because of excessive dilution of EtBr used.
5. Running buffer solution was stored for future use.

6th October, 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.

1. A concentration gradient of one of the strands was decided to be run in order to find out the minimum detectable mass of DNA.
2. Buffer pH was quantified
3. Another PAGE gel was cast.
4. Samples loaded in different lanes - B2(2,4,8,16 μl)(S2 stock), I1(S2 stock), L1(S2 stock), I1L1 annealed.
5. The gel electrophoresis was carried out at 70V in cold room.

Gel 1 - Lanes are 1 : B2(2uL), 2 : B2(4uL), 3 : B2(8uL), 4 : B2(16uL), 5 : I1, 6 : L1, 7 : I1L1 annealed
The lane for I1L1 shows a band higher than I1 and L1 lanes. However, both bands for I1 and L1 can not be at the same level.

This time at least 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. This caused the whole gel to become blue. Hence, more gels need to be run.

20th October, 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 up to 10ml.

1. A gel was run to confirm formation of our final structure and to check the correctness of strand design (it must not result in non-specific binding of I1, I2 to B). The percentage was decided to be 15 as in the previous gel, the bands stayed very near the top.
2. This time the lanes were loaded with following samples: I1(S2 stock), L1(S2 stock), I1L1 annealed, BI1I2 annealed, BL2 annealed, L2I2 annealed, BL2I2 annealed, mixture of BL2I2 annealed & I1L1 annealed.
3. The electrophoresis tank was filled with pre-chilled buffer.
4. Later,the gel was run at 70V for about half an hour in the cold room.
5. This caused the lanes in the center to move faster compared to the lanes on the side of the gel, possibly due to heat generation.
6. Then, the voltage was lowered to 60V for half an hour.
7. Finally, it was kept at 50V for one more hour.
8. 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.

Gel 2 - Lanes are 1 : I2L2 annealed, 2 : L2B annealed, 3 : I2L2B annealed, 4 : BI1I2 annealed, 5 : I2L2B annealed + I1L1 annealed, 6 : I1, 7 : L1, 8 : I1L1 annealed.
The lane for BI1I2 shows multiple bands, indicating the absence of secondary structures.
The band for I1L1 is significantly higher than L1 and I1. The difference between I1 and L1 lanes can be seen.

Things to be done:

1. Anneal a fresh set of samples
2. Prepare a 12% gel next time
3. Keep the running voltage low - Check Sambrook for the exact value (6V/cm of the electrode??)
4. Run a 10bp ladder along with the samples so that it will enable us for easy comparison with controls and will tell us whether gel running voltage is wrong or the sample preparation is wrong.

22nd October, 2013

The following samples were annealed - I1L1, BI1I2, BL2I2, BL2, BI2 for which 5uL of respective 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).

24th October, 2013

A 12% PAGE gel was prepared
4ml 29:1 acrylamide solution
2ml 5X TBE Buffer
0.7ml APS
3.3ml ddH20
10uL TEMED

1. Samples loaded were - I1, L1, I1L1 annealed, BI1I2 annealed, BL2 annealed, L2I2 annealed, BL2I2 annealed, BL2I2 annealed + I1L1 annealed
2. The gel was run at 50V constantly for 2 hours
3. The gel on staining showed that more time had to be given for running (many bands were at same level). Still no clue about the non-linearity of bromophenol blue line.

To be done

1. Load 2 same samples at starting and ending lanes to check effect of non-linearity of bromophenol blue band