Team:IISER Bhopal/Experiment

iGEM IISER Bhopal

Protocols

Super Competent Cell Preparation

  1. An isolated colony was taken from the plate, inoculated in LB media, and grown overnight at 37°C 200 rpm till OD600 reached 0.6. This was the Primary culture.
  2. The culture grown overnight was diluted to 1:100 in 50 mL SOB medium (i.e 500 μL of culture in 50 mL of SOB). This was the Secondary culture.
  3. Cells were grown at 18°C until the OD600 reached 0.4 approximately.
  4. The culture was chilled in ice for 30 mins.
  5. The culture was distributed into two chilled falcon tubes of 25 mL each.
  6. Cells were pelleted at 5000 rpm at 4°C for 10 mins and the supernatant was discarded.
  7. The cells were resuspended in 12.5 mL of ice cold Transformation Buffer (TB).
  8. They were kept in ice for 45 mins.
  9. Later, the cells were pelleted at 4000 rpm at 4°C for 10 mins and the supernatant was discarded.
  10. The pellet was dissolved in 2.5 mL ice cold TB with 175 μL of DMSO.
  11. 100 μL of this suspension was transferred into 1.5 mL microcentrifuge tubes.
  12. The tubes were Flash frozen using liquid Nitrogen in a benchtop Liquid Nitrogen container and stored at -80°C.

Tips:

  • Chilled centrifuge tubes were used to allow proper maintenance of temperature.
  • DMSO was filter sterilized to avoid contamination.
  • The colony used to make the primary culture was picked from a freshly prepared plate.
  • Supernatant was completely discarded to avoid remains of media while making sure the pellet was not lost.
  • Liquid Nitrogen was handled with caution to avoid accidents and cryogenic gloves were worn at all times.

Efficiency of competent cells:

Number of colonies were counted on a light field or a dark background, such as a lab bench. The following equation was used to calculate the competent cell efficiency. If done in triplicates, the average cell colony was calculated.

  • Efficiency (in cfu/µg) = [colonies on plate (cfu) / Amount of DNA plated (ng)] x 1000 (ng/µg)
  • Amount of DNA plated (ng) = Volume DNA added (1 µL) x concentration of DNA (refer to vial, convert to ng/µL) x [volume plated (100 µL) / total reaction volume (1000 µL)]

The above protocol was obtained from Cold Spring Harbour Laboratory.

Plasmid Transformation

  1. Competent cells were thawed on ice: This may take 10-15 mins for a 260 µL stock. The unused competent cells were discarded. Unused thawed cells are not to be refrozen, as this would drastically reduce transformation efficiency.
  2. 50 µL of competent cells were pipetted into 1.5 mL microcentrifuge tube: 50 µL in a 1.5 mL tube per transformation. Tubes were labelled, pre-chilled, and were kept in a floating tube rack for support. All tubes were kept on ice. A tube was included for the control.
  3. 1 µL of resuspended DNA was pipetted into 1.5 mL tube: Plasmid was pipetted into appropriately labeled tube and gently mixed. All tubes were kept on ice.
  4. 1 µL of control DNA was pipetted into 2 mL tube: 1 μL of 10 pg/μL control was pipetted into the control transformation mix and gently pipette up and down a few times. All tubes were kept on ice.
  5. The tubes were incubated on ice for 30 mins: Tubes were gently agitated/flicked to mix solution, but returned to ice immediately.
  6. The tubes were given a heat shock at 42°C for 45 sec: 1.5 mL tubes were kept in a floating foam tube rack. They were placed in water bath, ensuring the bottoms of the tubes were submerged. Timing is critical.
  7. Incubated on ice for 5 mins: Transformation tubes were returned to the ice bucket.
  8. 950 µL SOC media was pipetted into each transformation mix: SOC should be stored at 4°C, but should be warmed to room temperature before use. The same was checked for contamination.
  9. They were incubated at 37°C for 1 hour, with shaking at 200-300 rpm.
  10. 100 µL of each transformation mix was pipetted onto petri plates and spread with sterilized spreader immediately. This helped ensure that a single colony could be picked.
  11. Cells were spun down at 6800 g for 3 mins and 800 µL supernatant was discarded. The cells were resuspended in the remaining 100 µL, and each transformation mix was pipetted onto petri plates and spread with sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.
  12. The plates were incubated overnight (14 to 18 hrs) at 37°C: If incubated for too long, colonies may overgrow and the antibiotics may start to break down leading to formation of satellite colonies.
  13. Single colonies were picked and confirmed for transformation using colony PCR.
  14. Number of colonies on control plate were counted and competent cell efficiency was calculated.
  15. Competent cells should have an efficiency of 1.5x108 to 6x108 cfu/µg DNA.

Polymerase Chain Reaction (PCR)

Component Quantity (For 50μL reaction)
Template DNA 10ng
5X HF Buffer 10μL (1X)
dNTP (2.5mM) 4μL
Forward primer(10μM) 1μL
Reverse primer(10μM) 1μL
MilliQ Make up to 50 μL
Phusion DNA polymerase 1 unit

Colony Polymerase Chain Reaction

  1. A cell lysis buffer was prepared and colonies were suspended in it (TE + 0.1% Triton-x100)
  2. Each colony to be screened was boiled in 15 µL lysis buffer for 5 mins.
  3. The contents were spun down for 10 mins at 13000 g.
  4. 2 µL of supernatant was used as a template for normal PCR reaction.
Component Quantity (For 50μL reaction)
Template DNA 10ng
5X HF Buffer 10μL (1X)
dNTP (2.5mM) 4μL
Forward primer(10μM) 1μL
Reverse primer(10μM) 1μL
MilliQ Make up to 50 μL
Phusion DNA polymerase 1 unit

Double Digestion

Component Quantity (For 50μL reaction)
DNA 1μg
5X DigestionF Buffer 5μL (1X)
Enzyme 1 1μL (10 units)
Enzyme 2 1μL (10 units)
Nuclease free water Make up to 50μL
  • The above reaction mixture was incubated at 37°C for 5-15 mins if both the enzymes have high fidelity else incubated overnight at 37°C.
  • It was made sure that the buffer was compatible with both the enzymes.

Ligation

Component Quantity (For 20μL reaction)
Insert DNA (1kb) 37.5ng
Vector DNA (4kb) 50ng (1X)
T4 DNA Ligase Buffer (10X) 2μL
Nuclease free water Make up to 20μL
T4 DNA Ligase 1μL
  1. The above reaction was set up in a microcentrifuge tube on ice.
  2. The reaction was gently mixed by pipetting up and down and microfuge briefly.
  3. For cohesive (sticky) ends, it was incubated at 16°C overnight or room temperature for 10 minutes.
  4. For blunt ends or single base overhangs, it was incubated at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
  5. The reaction was heat inactivated at 65°C for 10 minutes.
  6. It was chilled on ice and 1-5 μL of the reaction was transformed into 50 μL competent cells.
  • T4 DNA Ligase was added last.
  • The molar ratio of 1:3 vector to insert for the indicated DNA sizes was used.
  • The T4 DNA Ligase Buffer was thawed and resuspended at room temperature.

Freeze-Thaw Cycle

  1. E. coli DH5α was grown in Luria Broth at 37ºC with shaking (220 rpm), overnight
  2. The culture was diluted (1:50) and grown till OD600 reached 0.5.
  3. The culture was diluted to 10-4 in LB (all dilutions were done in triplicate).
  4. Final culture dilution made was to 10-5 in LB.
  5. 100uL of each dilution was plated on LB petri dishes and grow overnight at 37 ºC. Number of colonies were counted on the next day.
  6. Final dilutions of 10-5 were kept at -20 ºC.
  7. After 48 hours,frozen dilutions were removed and thawed at room temperature.
  8. 100 μL of the dilutions was plated on LB agar plates and grown overnight at 37 ºC. Number of colonies were counted on the next day.


Growth Cycle

  1. A primary culture was set and grown at 37 °C until it reached saturation in an incubator-shaker at 200 rpm.
  2. A secondary culture was grown at 37°C until OD600 reaches 0.2 in an incubator- shaker at 200 rpm.
  3. The secondary culture was induced with 100 µM IPTG (with the following variants 0 µM, 10 µM, 15 µM, 20 µM, 30 µM, 50 µM, 100 µM, 500 µM, 1000 µM) in a 96 well plate with appropriate controls ( uninduced with 0 µM of IPTG and blank with LB + ampicillin only)
  4. Absorbance was noted at 600 nm every hour till OD600 reached saturation at different temp in an incubator-shaker
  5. The data obtained was analysed and plotted as appropriate graphs.

SDS PAGE:

  • A 12% separation gel was prepared by mixing the following reagents in the given order.
    • Reagent Quantity
    Acrylamide/bisacrylamide 4mL
    SDS 100uL
    Tris base (pH=8.8) 2.6mL
    TEMED 10uL
    APS (10%) 100uL
    H2O 3.2mL
  • The gel was poured, leaving ∼2 cm below the bottom of the comb for the stacking gel.
  • The top layer was washed with isopropanol and the gel was left to set for 30 minutes.
  • Isopropanol was washed with distilled water.
  • Stacking gel (4%) was prepared using the following reagents and mixing them in the given order.
    • Reagent Quantity
    Acrylamide/bisacrylamide 1.3mL
    SDS 100uL
    Tris HCl (pH 6.8) 2.5mL
    TEMED 10uL
    APS (10%) 100uL
    H2O 6.1mL
  • Stacking gel was poured on top of the running gel.
  • Comb was added to the stacking gels to make wells.
  • The gel was clamped into the apparatus and both the chambers were filled with running buffer.
  • Samples were loaded along with a molecular mass protein marker for separation by electrophoresis.

The preparation of standard solutions/buffers is listed in the pdf listed below:

Protocol