Team:IISER-Pune-India/Protocols

Protocols

Media Preparation

LB liquid medium: 1000mL

Requirements

  • 1L autoclave bottle or erlenmeyer flask
  • LB broth powder
  • Distilled or milliQ water
  • Magnetic stirrer and bead(optional)
  • Measuring cylinder
  • Autoclave tape
  • Cotton plug(for the flask)
  • Aluminium foil
  • Autoclaving machine

Procedure

  1. In a 1L autoclave bottle or flask , add 25g LB broth powder.
  2. Add distilled water to make the solution upto 1000mL.
  3. Either Swirl or use magnetic stirrer and a bead to mix (might not dissolve completely).
  4. Make a nice cotton plug by rolling and folding a big piece of cotton and put it on the mouth of the flask. (Try pulling it out once, if it makes a noise then ,Bravo!)
  5. Put an Aluminium foil on top of it and a piece of autoclave tape
  6. Keep the cap slightly open if using a bottle.
  7. Autoclave at 121°C
  8. Cool to RT before use. Do not tighten cap until cool.
LB agar plates: (~45-50 plates)

Requirements

  • 2L Erlenmeyer flask
  • LB broth powder
  • Bacteriological Agar
  • Distilled or milliQ water
  • Magnetic stirrer and bead(optional)
  • Measuring cylinder
  • Autoclave tape
  • Cotton plug(for the flask)
  • Aluminium foil
  • Autoclaving machine
  • Petri plates

Procedure

  1. Prepare 1L of LB liquid (25g broth powder per 1L) in a 2000mL Erlenmeyer flask.
  2. Add 15g (1.5%) bacteriological agar
  3. Use magnetic stirrer and bead to mix , if not available simply swirl to mix. Powder will not dissolve completely.
  4. Make a nice cotton plug by rolling and folding a big piece of cotton and put it on the mouth of the flask(Try pulling it out once, if it makes a noise then, Bravo!)
  5. Add a fresh piece of aluminum foil to cover the top, and add a fresh piece of autoclave tape.
  6. Autoclave at 121°C
  7. After cycle is complete, remove to benchtop to cool to ~50 C. (As a ballpark, this is still quite warm, but cool enough to leave your gloved hand on the outside of the flask for several seconds without burning it. The media should still be liquid but should be just beginning to thicken)
  8. If you are adding antibiotics, add them at this point, and swirl the flask vigorously for several seconds to mix (1000 times dilution of the stock solution)
  9. Set out your plates, and label the bottom with the appropriate antibiotic: A=ampicillin, C=chloramphenicol, K=kanamycin, T=tetracycline, S=streptomycin
  10. Pour out the media into petri dishes as follows: Remove the lid to a dish, and remove the foil cover to the flask. Pour just enough LB agar into the petri dish to completely cover the bottom of the dish, then replace the lid.
  11. Finish pouring dishes within 5 minutes of adding the antibiotic.
  12. Dishes should be stored UPSIDE DOWN in the cold room.
SOB medium: (250mL)

Requirements

  • 1L autoclave bottle or erlenmeyer flask
  • 6.25g LB broth powder
  • 2.5mM KCl
  • 20mM MgSO 4 .7H 2 O
  • Distilled or milliQ water
  • Magnetic stirrer and bead(optional)
  • Measuring cylinder
  • Autoclave tape
  • Cotton plug(for the flask)
  • Aluminium foil
  • Autoclaving machine

Procedure

  1. In a 1L autoclave bottle or flask , add 6.25g LB broth powder.
  2. Calculate and weigh 2.5mM KCl and 20mM MgSO 4 .7H 2 O.
  3. Adjust pH to 7.5(using NaOH)
  4. Add distilled water to make the solution upto 250mL.
  5. Either Swirl or use magnetic stirrer and a bead to mix(might not dissolve completely) .
  6. Make a nice cotton plug by rolling and folding a big piece of cotton and put it on the mouth of the flask.(Try pulling it out once, if it makes a noise then, Bravo!)
  7. Put an Aluminium foil on top of it and a piece of autoclave tape
  8. Keep the cap slightly open if using a bottle.
  9. Autoclave at 121°C
  10. Cool to RT before use. Do not tighten cap until cool.

Cloning

2A Assembly

Requirements

  • Your part samples, A: Miniprepped DNA or gblock
  • Linearized Plasmid Backbone (with a different resistance to the plasmid backbones containing your part samples)
  • EcoRI, XbaI, SpeI, PstI, DpnI
  • NEB Buffer 3.1
  • dH2O

Digestion

Enzyme Master Mix for Plasmid Backbone (10ul total) Enzyme Master Mix for Part A (10ul total)
  • 1ul 1X NEB Buffer 3.1
  • 0.2 ul EcoRI
  • 0.2 ul PstI
  • 0.2 ul DpnI (Used to digest any template DNA from production)
  • Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)
  • 4.7ul dH2O
  • 1 ul 1X NEB Buffer 3.1
  • 0.2 ul EcoRI
  • 0.2 ul PstI
  • Add 100ng Part A
  • Add dH2O upto 10ul
  • Digest all three reactions at 37 ℃ /4 hours, heat kill 80 ℃ /20 min

Ligation

  • Add 2ul of digested Plasmid Backbone (25 ng)
  • Add 3X molar amount of Part A (EcoRI-PstI digested) fragment (< 3 ul)
  • Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase
  • Add 0.5 ul T4 DNA ligase
  • Add water to 10 ul
  • Ligate 16 ℃ /overnight, heat kill 80 ℃ /20 min
  • Transform with 1ul of product
3A Assembly

Requirements

  • Your two part samples, A and B: Miniprepped DNA or gblock
  • Linearized Plasmid Backbone (with a different resistance to the plasmid backbones containing your part samples)
  • EcoRI, XbaI, SpeI, PstI, DpnI
  • NEB Buffer 2.1, Buffer 3.1
  • dH2O
  • Digestion

    Enzyme Master Mix for Plasmid Backbone (10ul total) Enzyme Master Mix for Part A (10ul total) Enzyme Master Mix for Part B (10ul)
    • 1ul 1X NEB Buffer 2.1
    • 0.2 ul EcoRI-HF
    • 0.2 ul PstI
    • 0.2 ul DpnI (Used to digest any template DNA from production)
    • Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)
    • 4.7ul dH2O
    • 1 ul 1X NEB Buffer 3.1
    • 0.2 ul EcoRI
    • 0.2 ul SpeI
    • Add 100ng Part A
    • Add dH2O upto 10ul
    • 1 ul 1X NEB Buffer 3.1
    • 0.2 ul XbaI
    • 0.2 ul PstI
    • Add 100ng Part A
    • Add dH2O upto 10ul
    • Digest all three reactions at 37 ℃ /4 hours, heat kill 80 ℃ /20 min

    Ligation

    • Add 2ul of digested Plasmid Backbone (25 ng)
    • Add equimolar amount of Part A (EcoRI SpeI digested) fragment (< 3 ul)
    • Add equimolar amount of Part B (XbaI PstI digested fragment) (< 3 ul)
    • Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase
    • Add 0.5 ul T4 DNA ligase
    • Add water to 10 ul
    • Ligate 16℃/overnight, heat kill 80℃/20 min
    • Transform with 1ul of product
RF-Cloning(Over-extension PCR)

Reaction volumes

pCAT template RBS template Final concentration
  • 10X Pfx buffer
  • 10mM dNTP
  • 50mM
  • Megaprimer
  • Template DNA
  • Pfx polymerase
  • dH2O
  • Total volume
  • 2μl
  • 0.6 μl
  • 0.4 μl
  • 4 μl
  • 2 μl
  • 0.8 μl
  • 10.2 μl
  • 20 μl
  • 2ul
  • 0.6 ul
  • 0.4 ul
  • 4 ul
  • 2 ul
  • 0.8 ul
  • 10.2 ul
  • 20 ul
  • 1X
  • 0.3mM
  • 1mM
  • -
  • -
  • 1U
  • 10X Pfx buffer

2-step PCR conditions

Process Temperature Time
Initial denaturation 94o 5'
30 cycles 95o 15-30s
68o 1 min/kb
Final extension 65-68o 5'
Hold 4o
  • Add 20 units of Dpn1
  • Incubate 2 hrs of at 37oC
  • Heat inactivate 80oC for 20 mins
Transformation

Requirements

  • Resuspended DNA to be transformed
  • 10pg/µl Positive transformation control DNA (e.g. pSB1C3 w/ BBa_J04450, RFP on high-copy chloramphenicol resistant plasmid.)
  • Competent Cells (50µl per sample)
  • 1.5mL Microtubes
  • SOC/LB Media (250µL per sample)
  • Petri plates w/ LB agar and antibiotic (2 per sample)

Procedure

  • Pipette out 1μl of the plasmid into an eppendorf.
    • Add 50μl competent cells into it.
    • Incubate the eppendorf in ice for 30 minutes.
  • Suscept it to a 42 ℃ heat shock for 60 sec using a water bath.
  • Now incubate this on ice for a short period of five minutes.
  • Add 250μl of LB/SOC to the eppendorf resulting in 300μl of the solution.
  • Incubate this solution at 37℃ for 2 hours.
  • Centrifuge the solution at 3000 rpm for 5 minutes and resuspend the pelette in 100μl of LB/SOC.
  • Plate the entire amount in the required antibiotic plate.
  • Incubate the plate at 37℃ overnight.
  • Count the number of colonies.
Bacterial Growth Curve in SOB media at 20℃

Requirements

  • 200mL Conical flasks(autoclaved at 121C)x2
  • Cuvettes
  • Spectrophotometer
  • Cotton plugs
  • 1mL micropipette
  • 1mL micropipette tips
  • Incubator
  • Bacterial culture (DH5a and MG1655)
  • SOB
  • Ethanol
  • Distilled water

Procedure

  • Preparation of SOB(iGEM protocol)
  • Inoculation
  • Initial OD
  • Inoculate 200ul of culture in 50mL SOB in conical flasks
  • Take OD measurement at Ohrs
  • Take OD measurements every hour
  • Plot OD VS Time graph
Ultra-competent cells

Procedure

  • Grow a single colony of MG1655/DH5α in 2mL LB/SOB @ 37℃ overnight
  • Inoculate 10mL SOB with 40µL of saturated above culture.
  • Incubate @ 20℃ 250-rpm overnight
  • Take it out of the incubator and keep the culture on ice [IMMERSE THE WHOLE STAND IN ICE]
    • Check OD to be around 0.3
    • Cool it on ice
    • Centrifuge @ 3000g (rcf) @4℃ for 10 minutes
    • Discard the supernatent
    • Resuspend it in 3mL ice cold CCMB80.
    • Incubate on ice for 20 minutes.
    • Centrifuge @3000g(rcf) @ 4℃ for 10 minutes
    • Discard supernatant.
    • Dissolve the pellet in 400µL ice cold CCMB80
    • Measure OD of the cell suspension
    • 50µL cells + 950µL SOB [Blank: 50µL CCMB80 + 950µL SOB]
    • Calculate the OD of cell suspension considering the dilution factor and measured OD.
    • Dilute the suspension so that final OD is between 1 and 1.5.
    • Aliquote 50µL in 0.6mL microcentrifuge tubes.
    • Incubate on ice for 20 minutes{flash freeze using liquid nitrogen}
    • Store @ -80℃ in cryo box labelled properly.

Experiments with T7 bacteriophage

Experiments with T7 bacteriophage

Experimental protocol

We infected E. coli with different dilutions of T7 bacteriophage. We followed the protocol below.

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