Biomod/2013/IIT-Madras/labnote: Difference between revisions

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* Nano-drop Spectrophotometer
* Nano-drop Spectrophotometer
* Gel apparatus (for Native PAGE)
* Gel apparatus (for [http://www.ncbi.nlm.nih.gov/pubmed/22585476 Native PAGE])
* Falcons, chemicals for Buffer preparation
* Falcon tubes


For Phase 2:
For Phase 2:
* Spectrofluorometer
* Spectrofluorometer
* Heat Blocks
* Heat Blocks
* Water Bath (if possible at T = 25°C)
* Water bath (at 25°C)
* Fluorescence Microscopy
* Fluorescence Microscopy
* AFM (If possible)
* [http://en.wikipedia.org/wiki/Atomic_force_microscopy AFM] (if slot available)




'''2nd September, 2013'''
'''2nd October, 2013'''


Materials used:
Materials used:


3 Falcon tubes(15 mL) - Used for preparing KCl stock & phosphate buffer <br />
3 Falcon tubes(15 mL) - Used for preparing KCl stock & phosphate buffer<br />
2 Falcon tubes(50 mL) - Used for ddH<sub>2</sub>O <br />
2 Falcon tubes(50 mL) - Used for ddH<sub>2</sub>O<br />
7 Eppendorf tubes(1.5 mL) - Used for S2 stock preparation <br />
7 Eppendorf tubes(1.5 mL) - Used for S2 stock preparation<br />
3 Eppendorf tubes(2 mL) - Used for Potassium Phosphate buffer preparation <br />
3 Eppendorf tubes(2 mL) - Used for Potassium Phosphate buffer preparation<br />


# ddH<sub>2</sub>O was added to all nucleotides in order to make their concentration up to 100 µM.  
 
# ddH<sub>2</sub>O was added to all nucleotides in order to make their concentration up to 100 µM. Call this stock S1.
# Stock S2, was prepared by diluting the above stock solutions by a factor of 100.
# 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
# All the stock solutions were labelled and kept in the Deep Freeze
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'''3rd September, 2013'''
'''3rd October, 2013'''


Materials used:  
Materials used:  
Line 43: Line 44:
5 Eppendorf Tubes (0.5ml)<br />
5 Eppendorf Tubes (0.5ml)<br />


# All the S2 stock solutions were quantified using NanoDrop spectrophotometer.
 
# 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
# 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
# Annealing mixtures were prepared - BI1I2, I1L1, BL2I2, L2I2, L2B by using the composition as given in the protocol in 0.5 ml Eppendorf Tubes
Line 50: Line 52:




'''4th September, 2013'''
'''4th October, 2013'''
 
Materials used:<br />
 
2 Eppendorf tubes(2ml)<br />
Conical flask(for preparation of gel running buffer)<br />
1 autoclaved Falcon tube(15ml)<br />
 
 
Reagent preparation:<br />
 
5X TAE buffer:<br />
1.63g Tris Base<br />
0.825g Boric Acid<br />
600µL of 0.5M EDTA solution<br />
 
 
5X TBE buffer:<br />
10.8g Tris Base<br />
5.5g Boric Acid<br />
4ml of 0.5M EDTA solution<br />
 
 
# 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.
# 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 October, 2013'''
 
Reagent preparation:
 
20% PAGE components:<br />
6.6ml of 29:1 acrylamide<br />
0.07ml of APS<br />
2ml of 5X TBE Buffer<br />
2.3μl of TEMED<br />
Appropriate amount of water was added to make the final volume upto 10ml.


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
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
# 5X Running Buffer was diluted using can water
1.63g Tris Base
# 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.
0.825g Boric Acid
# EtBr stock solution (10mg/ml) was prepared by weighing 4.7 mg of EtBr.
600 µL 0.5M EDTA solution
# 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.
# Running buffer solution was stored for future use.


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
'''6th October, 2013'''


Gel Running Buffer was prepared - 200ml 5X TBE buffer
Reagent preparation:
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
Gel Running Buffer/5X TBE buffer:<br />
10.8g Tris Base<br />
5.5g Boric Acid<br />
4ml 0.5M EDTA solution<br />
Appropriate amount of water was added to make the final volume upto 200ml.


The buffer was stored in a conical flask


20% PAGE components:<br />
6.6ml of 29:1 acrylamide<br />
0.07ml of APS<br />
2ml of 5X TBE Buffer<br />
2.3ul of TEMED<br />
Appropriate amount of water was added to make the final volume upto 10ml.


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


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.
[[Image:GelPic-7-10.png|center|frame|'''Gel 1''' - Lanes are 1 : B2(2uL), 2 : B2(4uL), 3 : B2(8uL), 4 : B2(16uL), 5 : I1, 6 : L1, 7 : I1L1 annealed <br/> 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. ]]


Buffer solution was diluted using can water
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.


The buffer used was stored for further usage


20% PAGE components
'''20th October, 2013'''
6.6ml of 29:1 acrylamide
0.07ml of APS
2ml of 5X TBE Buffer
2.3ul of TEMED
Water to make upto 10ml


Reagent preparation:


'''6th September 2013'''
15% PAGE components:<br />
5ml of 29:1 acrylamide<br />
Gel Running Buffer was prepared - 200ml 5X TBE buffer
0.07ml of APS<br />
10.8g Tris Base
2ml of 5X TBE Buffer<br />
5.5g Boric Acid
10μl of TEMED<br />
4ml 0.5M EDTA solution
Appropriate amount of water was added to make the final volume up to 10ml.


Buffer pH was quantified


Another PAGE gel was cast .
# 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.
PAGE components
# 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.
6.6ml of 29:1 acrylamide
# The electrophoresis tank was filled with pre-chilled buffer.  
0.07ml of APS
# Later,the gel was run at 70V for about half an hour in the cold room.
2ml of 5X TBE Buffer
# 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.
2.3ul of TEMED
# Then, the voltage was lowered to 60V for half an hour.
Water to make upto 10ml
# 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.


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
[[Image:GelPic-20-10.png|center|frame|'''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. <br/>The lane for BI1I2 shows multiple bands, indicating the absence of secondary structures. <br/>The band for I1L1 is significantly higher than L1 and I1. The difference between I1 and L1 lanes can be seen.]]
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
Things to be done:<br />
5ml of 29:1 acrylamide
#Anneal a fresh set of samples
0.07ml of APS
#Prepare a 12% gel next time
2ml of 5X TBE Buffer
#Keep the running voltage low - Check Sambrook for the exact value (6V/cm of the electrode??)
10ul of TEMED
#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.
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.
'''22nd October, 2013'''


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


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.
'''24th October, 2013'''


Things to be done
A 12% PAGE gel was prepared<br />
Anneal a fresh set of samples
4ml 29:1 acrylamide solution<br />
Prepare a 12% gel next time
2ml 5X TBE Buffer<br />
Keep the running voltage low - Check Sambrook for the exact value (6V per cm of the electrode ??)
0.7ml APS<br />
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.  
3.3ml ddH20<br />
10uL TEMED<br />




'''22nd September, 2013'''
#Samples loaded were - I1, L1, I1L1 annealed, BI1I2 annealed, BL2 annealed, L2I2 annealed, BL2I2 annealed, BL2I2 annealed + I1L1 annealed
#The gel was run at 50V constantly for 2 hours
#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.


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
To be done<br/>
#Load 2 same samples at starting and ending lanes to check effect of non-linearity of bromophenol blue band

Latest revision as of 22:29, 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 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
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