Team:IISc-Bangalore/Protocols

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).
  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. 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 chilled microfuge tube.
  4. Keep it in ice for 20 minutes.
  5. Spin at 5000 rpm for 5 mins at 4ºC. Discard supernatant and the remnants by tappng the inverted tube on tissue paper.
  6. Resuspend the pellet in 1 ml of chilled 10% glycerol
  7. Spin at 5000 rpm for 5 mins at 4ºC. Discard supernaatant and the remnants by tappng the inverted tube on tissue paper.
  8. Repeat step 6 and 7 two more times
  9. Resuspend the pellet in 40 ul of chilled 10% glycerol.
  10. Keep the cells in ice.
  11. Use the cells for electroporation 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.
  1. Thaw competent cells on ice.
  2. Pipette 50 μl of electrocompetent cells into a microfuge tube.
  3. Add 1 μl of DNA into the tube and mix it.
  4. Transfer the mixture to a chilled electroporation cuvette (washed with ethanol).
  5. Place it in the electroporator and give an electric pulse of appropriate voltage(1.8kV for small plasmids; 2.2kV for T4 DNA) and pulse time (4-6 μs)
  6. Add 1 ml SOC media into the cuvette immediately after the pulse. (Delay in adding SOC may reduce efficiency).
  7. Transfer the contents of the cuvette into a microfuge tube
  8. Incubate at 37°C 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.

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.
  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.
  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.
  1. NOTE: This protocol is to be used after the presence of insoluble protein in the form of inclusion bodies has been verified. This can be done by using lysis buffers with increasing concentrations of a detergent like SDS to see if the protein is recovered in the lysate (see Characterization for BBa_K2609007). To regain activity after urea denaturation, a refolding step is also needed.
  2. Pellet the cells after induction by centrifuging at 6,000 rpm for 10 mins (4ºC).
  3. Wash the cell pellet with 1xPBS (1:10 initial culture volume) twice followed by centrifugation at 6,000rpm (10mins at 4ºC).
  4. Sonicate the cells for 20 minutes (total) after resuspending them in 1/10 culture volume of 1xPBS with a 10 sec ON, 10 sec OFF pulse.
  5. Centrifuge at 13,000rpm for 15 min (4ºC)
  6. Resuspend the pellet in 8M Urea denaturation buffer (1:10 culture vol.) and incubate at 4ºC for 24 hrs with constant mixing.
  7. Centrifuge at 13,000rpm for 15 mins at 4ºC and store the supernatant.
  8. The supernatant should now contain your protein along with other cellular proteins. Use the following protocol to purify and refold it (provided it is 6xHis tagged).
  1. The protein lysate from the last protocol can be directly used for 6xHis purification with the following buffer compositions: Binding buffer, Wash Buffer and Elution buffer.
  2. To refold the protein after purification, dialyse against the following buffers in a step by step fashion (constant stirring, 4ºC, 10 hrs per buffer)
    1. 6M Urea buffer
    2. 4M Urea buffer
    3. 2M Urea buffer
    4. 0M Urea buffer

Phage Work

  1. Make 1% secondary inoculum(100ml) of the MG1655 (the strain of E.coliwe used)
  2. Grow with aeration till 0.6 OD.
  3. Add 1 ml of phages(109PFU/ml) into the culture.
  4. Incubate it at 37ºC till the culture gets cleared.
  5. Centriguge the lysate at 4000g for 20 minutes.
  6. Collect supernatant into a sterile falcon tube.
  7. Filter sterilize using 0.22 um filter to yield lysate.
  8. Add 1/10th volume of chloroform to the supernatant, vortex and incubate at room temperature for 10 minutes.
  9. Centrifuge at 4000g for 5 minutes.
  10. Store at 4ºC
  1. Make 1% secondary inoculum of the MG1655 (the strain of E.coli we used)
  2. Grow with aeration till 0.6 OD.
  3. Add 50 μl of the culture into a sterile falcon tube.
  4. Add 50 μl of phages into the tube and mix it.
  5. Leave it for 20-30 minutes.
  6. Add 3 ml of top agar into the tube and mix it.
  7. Pour it into a LB plate immediately and wait till the top agar soldifies.
  8. Incubate the plate for 16-18 hours.
  9. You will see a lawn of bacteria (if your phages did not infect the cells) or you will see plaques or clearings (if the phages infected the cells). Count the number of plaques and you will get the PFU/ml(plaques forming units) value for your phage units.

We used this assay to relate the MOI of bacteriophages with the MIC of a particular antibiotic, with respect to a fixed volume of bacterial culture. This assay essentially gives the viability of cells.

  1. Prepare a bacterial inoculum identical to the one in step 3 of the PFU determination assay.
  2. Aliquot the following into 1.5ml microfuge tubes: 470ul LB medium, 25ul of the above bacterial inoculum and 5ul of a particular phage/antibiotic dilution. Make sure to include controls in which there are no bacteriophages or viruses, and one with only the medium. Create replicates of each tube.(Note: The solution above only contains 1% of the particular phage/antibiotic dilution and hence the concentration in the solution will be 10-2 times that in the prepared stock.)
  3. Vortex the tubes gently to mix the contents, and then proceed to shake-incubate them at 37° C and 220 RPM for two and half hours.
  4. Remove the tubes from the incubator and pipette out 100ul of each sample into a designated well in the microtitre plate. Make sure to label the wells appropriately and arrange the dilutions in an orderly manner. Also, include a positive control with only the cells.
  5. Pipette 20ul of well-mixed 0.02% resazurin solution (also called alamar blue) into each well. Use a resuspension-like action to ensure mixing with the sample. Do this procedure in a darkened environment.
  6. Cover the plate and wrap it in aluminium foil. Incubate it at 37° C for 20 minutes.
  7. Take out the plate, unwrap it and place it in the plate reader. Take absorbance readings for both 570nm and 600nm wavelengths of light.
  8. The percentage of dead cells is given by the formula, ((O2*A1)-(O1*A2))/((O2*P1)-(O1*P2)), where
    • O1 = molar extinction coefficient (E) of oxidized alamarBlue (Blue) at 570 nm = 80586
    • O2= E of oxidized alamarBlue at 600 nm = 117216
    • A1 = absorbance of test wells at 570 nm
    • A2 = absorbance of test wells at 600 nm
    • P1 = absorbance of positive growth control well (cells plus alamarBlue but no test agent) at 570 nm
    • P2 = absorbance of positive growth control well (cells plus alamarBlue but no test agent at 600 nm
  1. Take 1 ml of phage lysate in a microfuge tube.
  2. Add 10 ul of 0.1 M MgSO4 to the tube (final conc. of 1mM).
  3. Add 20 ul of 20 mg/ml lysozyme.
  4. Add 2 ul each of 20 mg/ml DNAse and 40 mg/ml RNAse.
  5. Incubate at 37°C for 2 hours.
  6. Add 20 ul of 0.1 M EDTA (final conc. of 2mM.
  7. Add 50 ul of 10% SDS solution (final conc. of 0.5 %)
  8. Add 3 ul of 20 mg/ml of Proteinase K (final conc. of 50 ug/ml).
  9. Incubate at 50°C for 1 hour.
  10. Add equal amount of equilibriated phenol:chloroform(1:1) mixture. Mix it well by rotating the tube slowly.
  11. Spin the tube at 13000g for 5 minutes. Pipette out the aqueous layer (usually the top layer) into another microfuge tube.Discard the rest.
  12. Add equal amount of chloroform to this tube. Mix