Team:IISc-Bangalore/Lab/Protocol

Protocols

The following is a list of all the general protocols we used during our laboratory hours.

DNA Work

Component Amound for 25µl Reaction
Phusion/Q5 Reaction Buffer (5x) 5 µl
10mM dNTPs 0.5 µl
10 µM Forward Primer 1 µl
10 µM Reverse Primer 1 µl
Template DNA 10pg to 50ng
Phusion/Q5 Polymerase 0.2 µl
ddH20 To 25 µl
Step Temperature Time Cycles
Initial Denaturation 98°C 2 min 1
Denaturation
Annealing
Extension		 98°C
60°C to 72°C
72°C		 20 sec
30 sec
30 sec/kb		 35-50
Final Extension 72°C 10 min 1
Component Amount for a 25 µl Reaction
Appropriate Buffer (10x) 2.5 µl
Substrate DNA 250ng to 5 µg
Restriction Enzyme 5 units/µg of plasmid DNA
1 unit/µg of linear dsDNA
ddH2O To 20 µl
  1. Incubate at 37°C for 1 to 2 hours.
  2. Deactivate at 80°C for 20 min.
Component Amount for a 10 µl Reaction
T4 DNA Ligase Buffer with ATP (10x) 1 µl
Vector Backbone 25-50ng
Insert DNA 25-100ng
(3:1 molar ratio of insert:vector)
T4 DNA Ligase 0.5 µl
ddH2O To 10 µl
  1. Incubate at 16°C overnight (10-12 hours).
  2. Deactivate at 80°C for 20 min.
  1. Get 1.5 mL culture in a 2 mL microfuge tube.
  2. Centrifuge at 5000 rpm for 5 min to get cell pellet.
  3. Add 1.5 mL culture and pellet repeatedly until 4.5 mL culture has been pelleted.
  4. Keep pellets on ice.
  5. Add 200 µL alkaline lysis solution I* to each microfuge tube, resuspend the solution using a micropipette and keep on ice for 5 min.
  6. Add 400 µL alkaline lysis solution II** to each microfuge tube, very gently invert, and keep on ice for 30 seconds.
  7. Add 300 µL alkaline lysis solution III*** to each microfuge tube, gently invert, and keep on ice for 1 min.
  8. Centrifuge at 13000 rpm for 5-10 min.
  9. Transfer supernatant to 2 mL microfuge tube.
  10. Add RNase A to supernatant (1 µL/mL).
  11. Keep at 37 degrees for 1 to 2 h.
  12. Add equal volume of chloroform.
  13. Centrifuge at 13000 rpm for 10 min (at 4 degrees).
  14. Take top aqueous layer (avoid white particles of protein).
  15. Add equal volume of chloroform.
  16. Centrifuge at 13000 rpm for 5 min (at 4 degrees).
  17. Take top aqueous layer (avoid white particles of protein).
  18. Add equal volume of isopropanol.
  19. Incubate on ice for 30 min 30.
  20. Centrifuge at 13000 rpm for 10 min.
  21. Decant supernatant (don’t let pellet fall!).
  22. Add 500 µL of 70% ethanol.
  23. Centrifuge at 13000 rpm for 10 min.
  24. Let ethanol dry up completely (pellet will turn invisible from white, don’t worry).
  25. Suspend in 10 µL of MilliQ (tried 20 µL later).


*Alkaline lysis Solution I

Glucose 50mM
Tris HCl (pH 8.0) 25mM
EDTA (pH 8.0) 10mM

**Alkaline lysis Solution II

NaOH 0.2 N
SDS 1% w/v

***Alkaline lysis Solution III

5 M Potassium acetate 60 ml
Glacial acetic acid 11.5 ml
ddH2O 28.5 ml

    We use Thermo Scientific GeneJET Gel Extraction Kit #K0691, #K0692
    IMPORTANT NOTES Prior to the initial use of the kit, dilute the Wash Buffer (concentrated) with ethanol (96-100%): 50 preps #K0691 250 preps #K0692 Wash Buffer (concentrated) 9 mL 45 mL Ethanol 45 mL 225 mL Total Volume 54 mL 270 mL After the ethanol has been added, mark the checkbox on the bottle to indicate the completed step. Examine the Binding Buffer for precipitates before each use. Re-dissolve any precipitate by warming the solution to 37 °C and cooling to 25 °C. Wear gloves when handling the Binding Buffer as this solution contains irritants.
    Do not reuse electrophoresis buffer when extracted DNA fragment will be used directly for sequencing.
    All purification steps should be carried out at room temperature. All centrifugation should be carried out in a table-top microcentrifuge at >12000 g (10 000-14 000 rpm, depending on the rotor type)
    Protocol A. DNA extraction from the gel using centrifuge Step Procedure
  1. Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 mL tube and weigh. Record the weight of the gel slice.
  2. Add 1:1 volume of Binding Buffer to the gel slice (volume: weight)
  3. Incubate the gel mixture at 50-60 °C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution, and mix. The color of the mix will become yellow.
  4. Transfer up to 800 µL of the solubilized gel solution (from step 3 or 4) to the GeneJET purification. column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
  5. Add 100 µL of Binding Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
  6. Add 700 µL of Wash Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
  7. Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.
  8. Transfer the GeneJET purification column into a clean 1.5 mL microcentrifuge tube. Add 50 µL of water at 60C to the center of the purification column membrane. Centrifuge for 1 min.
  9. Discard the GeneJET purification column and store the purified DNA at -20 °C.
  1. Inoculate 1% inoculum from overnight culture (i.e., 0.5 ml for 50 ml medium).
  2. Grow with aeration till 0.6 O.D. (600nm).
  3. Pipette 1 ml of the bacterial culture at 0.6 O.D. into a sterile microfuge tube.
  4. Chill the cells on ice for 5 min.
  5. Spin at 5000 rpm for 10 min at 4ºC. Discard supernatant by decanting and the remnants by tapping the inverted tube on tissue paper.
  6. Resuspend the pellet in 200 µl of ice cold solution I (0.1M MgCl2)
  7. Keep on ice for 30 min.
  8. Spin at 5000 rpm for 10 min at 4ºC.
  9. Resuspend the pellet in 50 µl of solution II (0.1M CaCl2 in 15% Glycerol)
  10. Keep the cells on ice for 1 h.
  11. Use the cells for transformation using heat shock or freeze the cells in liquid nitrogen and store at –70ºC for future use.
  1. Thaw competent cells on ice.
  2. Pipette 50uL of competent cells into 2mL tube.
  3. Pipette 1uL of resuspended DNA into 2mL tube.
  4. Pipette 1uL of control DNA into another 2mL tube (control).
  5. Heat shock tubes at 42ºC for 1 min.
  6. Incubate on ice for 5 min.
  7. Pipette 1ml SOC media to each transformation tube.
  8. Incubate at 37C for 1-2 hours, shaker recommended.
  9. Spin at 5000 rpm for 5 minutes and resuspend the pellet in 100 ul of SOC media .
  10. Spread it in a LB Agar plate with proper antibiotics using a spreader.

Super Optimal Broth with Catabolite repression (SOC) Medium

Yeast extract 0.5 %
Tryptone 2 %
NaCl 10 mM
KCl 2.5mM
MgCl2 10 mM
MgSO4 10 mM
Glucose* 20mM

* Add filter sterilized glucose after autoclaving the media with rest of the ingredients

Protein Work

  1. Take 50 ul of the sample in a microfuge tube.
  2. Add equal amount of SDS sample buffer* into it.
  3. Denature the protein by heating the tube in a 95ºC waterbath for 15-20 minutes.
  4. Load the sample into the SDS PAGE gel.
Recipe for stacking gel (5 ml)

Stacking buffer 0.63 ml
30% acrylamide 0.83 ml
Water 3.4 ml
10 % SDS 50 μl
10 % APS 50 μl
TEMED 10 μl

Add TEMED and APS just before pouring the soultion into the cast

Recipe for 12 % resolving gel (25 ml)

Resolving buffer 6.3 ml
30% acrylamide 10 ml
Water 8.2 ml
10 % SDS 250 μl
10 % APS 250 μl
TEMED 15 μl

Add TEMED and APS just before pouring the soultion into the cast


Sodium dodecyl sulphate sample buffer

Stacking buffer 8 ml
50 % glycerol 16 ml
10 % SDS 8 ml
DTT 960 mg
10 % SDS 8 ml
Bromophenol blue Enough to make it blue

  1. Inoculate 100 ml of 1% secondary inoculum from overnight culture .
  2. Grow with aeration till 0.6 O.D. (600nm).
  3. Pipette out 1 ml from the secondary inoculum into a microfuge tube and label it as uninduced sample.
  4. Add IPTG to the secondary inoculum to final concentration of 500 uM.
  5. Grow both induced and uninduced culture at 37ºC for 3 hours or 25ºC for 16 hours.
  6. Transfer the culture into centrifuge tubes.
  7. Centrifuge at 6000 rpm at 4ºC for 15 mins. Remove supernatant and resuspend it in 10 ml of protein lysis solution .
  8. Sonicate for 20 minutes (with 5 sec on, 10 sec off pulse).
  9. Centrifuge at 13000 rpm at 4ºC for 20 minutes. Take out the supernatant which contains the required protein.
  10. Run a SDS PAGE with the above supernatant to check for the protein.

Protein lysis solution


PMSF 1mM
Imidazole 15 mM
Lysozyme 0.5 mg/ml

The solution is made in PBS


  1. Grind fresh, desiccated ammonium sulphate salt using a mortar and pestle until it is reduced to a fine powder.
  2. Place the protein lysate (100 ml) on a magnetic stirrer at 4 ̊C. Ensure that the temperature doesn’t go beyond 4 ̊C throughout the course of the experiment.
  3. Very slowly add the ground ammonium sulphate powder to the protein lysate (with continued stirring) to a the concentration of 10% saturation of the salt.
  4. Keep the lysate at 4 ̊C with constant stirring for 30 minutes to allow the precipitate to form.
  5. Centrifuge the solution at 20,000 rpm at 4 ̊C for 20 minutes.
  6. Pour the supernatant back into a Duran bottle and resuspend the pellet containing the precipitated proteins in 3-5 ml of PBS.
  7. Store the resuspended protein precipitated at that concentration (10%) in a falcon tube & store at -20 ̊C.
  8. Use the supernatant as the new lysate and set it up for magnetic stirring at 4 ̊C.
  9. Now very slowly add ammonium sulphate powder to reach a final concentration of 20% from the initial 10% of the supernatant.
  10. Repeat the same steps of protein precipitation and centrifugation, while increasing the ammonium sulphate concentration by 10% at each step.
  11. Continue the procedure until 90% -100% saturation of ammonium sulphate is reached.
  12. Run an SDS PAGE with the proteins precipitated at different saturation levels of ammonium sulphate.
  13. The concentration at which the protein of interest gives the desired band is the concentration at which it precipitates.
    14. Note:
    The amount of Ammonium Sulphate to be added at each saturation step can be calculated using the Ammonium Sulphate Calculator by EnCor Bio: http://www.encorbio.com/protocols/AM-SO4.htm
  1. Prepare the cell cultures at the desired growth stage.
  2. Take the cell cultures in 2 ml eppendorfs.
  3. Centrifuge the culture at 6000 rpm at 4ºC for 15 mins
  4. Discard the supernatant. Add the protein lysis solution to the pellet, to get solution with protein aggregates.
  5. Sonicate the cells for 20 minutes (with 5 sec on, 10 sec off pulse) to get the cell lysate.
  6. Perform the column chromatography with the supernatant after centrifugation.
  7. Wash the crude protein lysate followed by elution.
  8. Collect the samples into eppendorfs.
  9. Run an SDS-PAGE to get an estimation of the purity and concentration of the protein obtained
  1. Add 500 ul of Ni-NTA beads to a microfuge tube.
  2. Add 1 ml of MQ water to it. Mix it for 15 minutes.
  3. Spin it at 5000 rpm at 4ºC for 5 minutes. Pipette out and discard the supernatant carefully without disturbing the beads.
  4. Add the cell lysate containing the required protein to the tube. Mix it for 30 hour. The Histagged protein would bind to the beads.
  5. Spin it at 5000 rpm at 4ºC for 5 minutes. Pipette out the supernatant into another microfuge tube and label it as supernatant after binding.
  6. Add 1 ml wash buffer into the tube. Mix it for 15 minutes.
  7. Spin it at 5000 rpm at 4ºC for 5 minutes. Pipette out the supernatant into another microfuge tube and label it as Wash.
  8. Add 1 ml elution buffer into the tube. Mix it for 30 minutes.
  9. Spin it at 5000 rpm at 4ºC for 5 minutes. Pipette out the supernatant into another microfuge tube and label it as Elution. This would contain the required His-tagged protein.
  10. Rin an SDS PAGE with the supernatant, wash and elution to check for the purified protein.

Wash Buffer

Tris 50 mM
NaCl 300 mM
Imidazole 40 mM

The pH should be adjusted to 7.8

Elution Buffer

Tris 50 mM
NaCl 300 mM
Imidazole 200 mM

The pH should be adjusted to 7.8


  1. Inoculate one colony of the transformed bacteria in a 5ml LB tube containing required antibiotics (Ampicillin and Gentamicin for pBAD and pJAT respectively) and 0.2% (w/v) Glucose.
  2. Incubate in Shaker until it reaches an OD of 0.6.
  3. Transfer 1 ml of LB into microfuge tube.
  4. Centrifuge at 6000 RPM for 5 min.
  5. Decant Supernatant and re-suspend pellets in 1 ml LB medium for LB wash.
  6. Repeat steps 4-5 two more times.
  7. Make 4 serial dilutions of 1/10 th concentrations in LB medium.
  8. Place a 2ul spot of all dilutions in a row on 7 different plates, containing different concentrations (w/v) of glucose and arabinose as follows: 0.2% glucose, 0.04% glucose, 0.007% glucose, 0% glucose/arabinose, 0.02% arabinose, 7x10-5% arabinose, 2x10-5% arabinose.
  9. Incubate the plates at 370 C for 12-16 hours.