Materials required

  • Electrophoresis apparatus
  • Micropipettes
  • Eppendorfs

Reagents required

  • Stock Buffer
  • TBE/TAE/TE- 10X / 50X
  • Agarose- 0.8% (W/V)
  • Ethidium bromide- 10mg/ml
  • Tracking Dye
  • Glycerol- 30%
  • Bromophenol blue- 0.025%

Preparation of agarose gel (0.8% concentration for 50ml)

For a gel platform of 10x7cm of thickness 0.6cm (~ 50 ml)

  1. Add 0.4g of agarose to a 50ml of 1X TBE (Working Buffer)
  2. Heat until the agarose dissolves.
  3. Add 5μl of ethidium bromide from a stock when the gel is about 56˚C (final concentration of 0.5μg/ml)

The volume of the well= well width x comb thickness x gel thickness gap


  1. Wash the gel platform thoroughly and wipe with tissue paper.
  2. Seal the open sides with adhesive tape.
  3. Place the comb on the gel tray in the appropriate place so that a gap of 0.5mm is left between the bottom of the comb's teeth and the floor of the gel platform.
  4. Pour the warm agarose solution into the gel tray and allow it to solidify.
  5. After the gel is completely solidified (30-45 min at room temperature), carefully remove the comb and untape, and mount the gel into the electrophoresis tank.
  6. Add just enough electrophoresis buffers to cover the gel to a depth of about 1mm.
  7. Connect the cords between the electrophoretic tank and the power pack.
  8. Mix the 5 μl of the sample with 5 μl of loading dye and load into the slots of the submerged gel using a disposable micropipette.
  9. Fix the initial voltage in the power pack at 50V for the first half an hour and subsequently increase the voltage to 150V after 30 minutes.
  10. Monitor the movement of DNA with loading dye and switch off the power pack before the dye with DNA reaches the lowest point of the gel.
  11. Monitor the resolved DNA under a UV Transilluminator at 260nm and infer the result.

Materials Needed

  • λ DNA or plasmid DNA
  • Restriction enzymes (EcoRI, BamHI)
  • 10X restriction enzyme buffer
  • Agarose
  • Gel loading buffer
  • Staining dye
  • 50X TAE buffer

Protocol for single digestion

1. Take a sterile 0.5ml microfuge tube

2. Add the following reagents in the order listed to a microfuge tube for a 20µl reaction

Single Digestion

Sterile DD Water 15µl
Restriction Buffer 2µl
DNA 2µl
Enzyme 1µl
Total 20µl

Double Digestion

Sterile DD Water 13µl
Restriction Buffer 2µl
DNA 3µl
Enzyme 1 1µl
Enzyme 2 1µl
Total 20µl

3. Mix it by pipetting and incubate the reaction at the appropriate temperature (37°C).

4. Run the restricted sample on a 1% agarose gel and observe the bands under the transilluminator.

Vector 3µl
Purified PCR fragment 4µl
5X Ligation Buffer 6µl
T4 DNA Ligase 1µl
NFW (Nuclease Free Water) Upto 29µl

Incubate @ 22°C for 1 hour. For maximum yield of useful recombinants, the reaction time can be extended to overnight @ 4°C.

PCR Reaction Mixture

Master Mix 12.5
Forward Primer 1.0
Reverse Primer 1.0
Template 2.0
Nuclease Free Water (NFW) 8.5
Total 25.0

Cyclic Conditions

The reaction mixture was denatured at 94°C for 5 min. Then PCR was run for 30 cycles at 94°C for 2 min, and annealing and extension temperature of 55°C for 1 min. The sample was then heated at 72°C for 1 min. Extension and final extension reaction are at 72°C for 7 mins. Finally, the product will be checked with 1% Agarose Gel electrophoresis with 1Kb DNA ladder. After electrophoresis, it is viewed under UV light.

Materials Required

  • Detergent-free, sterile glassware, and plasticware
  • Table-top OD600nm spectrophotometer
  • SOB
  • CCMB Buffer


  1. Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3. Use the "cell culture" function on the Nanodrop to determine OD value. OD value = 600nm Abs reading x 10.
  2. Fill an ice bucket halfway with ice. Use the ice to pre-chill as many flat-bottom centrifuge bottles as needed.
  3. Transfer the culture to the flat-bottom centrifuge tubes. Weigh and balance the tubes using a scale.
  4. Centrifuge at 3000g at 4°C for 10 minutes in a flat-bottom centrifuge bottle.
  5. Decant supernatant into the waste receptacle and bleach before pouring down the drain.
  6. Gently resuspend in 80 ml of ice-cold CCMB80 buffer.
  7. Incubate on ice for 20 minutes.
  8. Centrifuge again at 3000g at 4°C. Decant supernatant into the waste receptacle, and bleach before pouring down the drain.
  9. Resuspend the cell pellet in 10 ml of ice-cold CCMB80 buffer.
  10. Use Nanodrop to measure the OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.
  11. Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
  12. Incubate on ice for 20 minutes. Prepare for aliquoting.
  13. Aliquot into chilled 2ml microcentrifuge tubes or 50 μl into chilled microtiter plates.
  14. Store at -80°C indefinitely.
  15. Test competence by measuring O.D.
  16. Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles.


  1. Prepare 50 μl of competent cells in Eppendorf tubes.
  2. Add 1 μl of plasmids to each Eppendorf tube containing competent cells.
  3. Mix the contents well and incubate on ice for 30 minutes.
  4. Provide heat shock by placing the tubes at 42ºC for 2 minutes. Immediately, snap-chill the tubes in ice and incubate for an additional 10 minutes.
  5. Add 200 μl of LB medium to each tube and incubate at 37ºC for 1-2 hours.
  6. After incubation, spread-plate the culture onto the respective antibiotic-containing plates.
  7. Incubate the plates for 48 hours at 37ºC and check for transformation efficiency.

Materials required

  • Elution buffer (0.5 M sodium acetate (pH 7) and 1 mM EDTA (pH 8))
  • 95% ethanol
  • 80% ethanol
  • TE buffer (10x) (pH 8.0)
  • Gel fragment containing the DNA


  1. Excise the region of the gel containing the required DNA band using a scalpel.
  2. Add elution buffer to the gel slice until the level of the buffer is a few mm above the level of the excised gel band.
  3. Heat the solution in a water bath at 65°C until the agarose completely melts.
  4. Fast-freeze by placing in a -80°C freezer for 10-15 minutes.
  5. Immediately thaw the solution by centrifuging for 10 minutes.
  6. The supernatant is transferred to a new tube.
  7. Add elution buffer again to the pellet and repeat steps 3-6.
  8. Accumulate the supernatants and add an equal volume of 1-butanol.
  9. Rock the mixture for 15 minutes to remove the EtBr completely from the gel.
  10. Discard the supernatant and repeat steps 8 and 9 2-3 times.


  1. The assay mixture contained 450 μL of 1 mM ABTS, 500 μL of 0.1 M acetate buffer (pH 4.0) along with 50 μL of enzyme at a temperature of 20 °C.
  2. This mixture was tilted in the cuvette before taking the absorbance reading.
  3. The complete reaction took place in the cuvette and nowhere else.
  4. The duration of the reaction was not more than 60 s.

pAlx - 96 Well Plate Reader Experiment

  • With this construct, we showed that the alx promoter and riboswitch are able to regulate mNeonGreen expression, when pH is shifted to pH 8 and above.
  • To do so, we let bacteria grow and then inoculated fresh media with adjusted pH (7, 8, and 8.5) to an OD600 of 0.2.
  • After 20 and 40 min, 1 ml of each culture was taken and diluted to the lowest OD600 of the three samples, and then fluorescence and OD600 were measured with the plate reader (n = 3).

pAsr - 96 Well Plate Reader Experiment

  • Asr promoter (Pasr) is reported to be induced under acidic conditions.
  • It can be used as a reporter when the medium turns acidic. We thus measure the fluorescence intensity over a short period of time.
  • We first incubated the bacteria to the log phase (about 2 hours) in Luria-Bertani (LB) medium.
  • We then centrifuged the broth and resuspended the pellet using M9 medium with different pH values (pH 4, 4.25, 4.5, 4.75, 5, 5.5, 6, and 7; the pH value was adjusted with 1M HCl).
  • We then incubated it in the 96 well plates and measured its fluorescence intensity (absorbance: 485 nm, excitation: 535 nm) every 3 minutes for 30 minutes.
  • The difference in fluorescence intensity can be observed within 30 minutes.