Team:Makerere University/protocols

Home page Wiki


Plasmid resuspension Protocol

Requirements:

  • Centrifuge
  • TE buffer or PCR water (30 – 40 µl)

    • Procedure:
    • We spun the microfuge tubes with lyophilized plasmid DNA at top speed so as to collect at the bottom.
    • We added 30 – 40 µL of TE buffer or PCR water mixed by pipetting up and down
    • We Stored the resuspended DNA at -200C.
    TRANSFORMATION
    Materials:
    • LB broth (100ml)
    • LB agar
    • Competent cells, E.coli BL21
    • Resuspended plasmid DNA (300ng/µl)
    • Ampicillin (100µg/ml)
    • Ice, incubator, centrifuge.

    Procedure:
    • We pipetted 100µl of competent E.coli BL21, with the plasmid DNA at a concentration of 300ng/ml in to a clean eppendorf tube and incubated on ice for 30 minutes.
    • Then immediately we incubated in a water bath at 420c for 90 seconds and assured proper timing.
    • We removed them from a water bath then placed them on ice for 2 minutes.
    • We added 1ml of LB medium containing ampicillin to each tube and placed them in an incubator at 370c for 45 minutes.
    • We plated cells on LB agar containing Ampicillin using a pipette dispensed 100µl of each cell culture ( for PETase and MHETase) to each petri dish with LB-AMP agar.
    • We incubated at 370C in an incubator overnight We sub cultured the overnight cell culture into the LB broth media containing ampicillin (100ug/ml) We prepared 50ml of LB-AMP broth using;
      • Yeast extract – 0.25g
      • Tryptone – 0.5g
      • Sodium chloride – 0.25g
      • Weighed into a clean glass bottles topped up with 50ml of distilled water, covered with a topper, and aluminum foil and autoclaved for 15 minutes at 1210C at 20Ψ Left the LB broth to cool down before addition of ampicillin, 50µl of ampicillin were added.
    Procedure for sub culturing
    • Measured 10ml of LB-AMP broth into two 50ml falcon tubes, picked 1-2 single colonies from each of the culture plates for the PETase and MHETase transformants.
    • Incubated at 370C for 6 hours at 250 rpm on a rotary incubator.
    Plasmid DNA isolation using alkaline lysis method
    Materials:
    • Alkaline solution 1, cell resuspension solution (10mM EDTA, 50mM Tris HCL, ph. 8.0 topped up with distilled water to 100ml)
    • Lysis solution (0.2M NaOH, 1% SDS )
    • Neutralization solution (0.55 M potassium acetate solution, pH. 4.8)
    • Isopropanol
    • 70% ethanol.
    Procedure
    • Poured overnight culture into 1.5 ml eppendorf tube
    • Centrifuged 4ml of the overnight culture at 14000rpm for 1 minute.
    • Removed supernatant from the tube, ensured that the pellet was dry.
    • Added 200µl of cell resuspension solution, then vortexed so as to resuspend the cells.
    • Added 250µl of cell lysis solution to the completely resuspended cells, mixed by inverting the tubes 4-6 times until the solution became viscous.
    • Added 300µl of neutralization solution and mixed by inverting the tube several times.
    • Centrifuged for 5 minutes at 14000 rpm.
    • Then transferred supernatant into two clean eppendorf tubes.
    • Added equal volume of isopropanol to the supernatant and mixed by inverting the tubes several times.
    • Incubated the tubes at -80oC for 30 minutes.
    • Centrifuged the tubes at 14000 rpm for 5 minutes.
    • Removed the supernatant and added 600µl of 70% ethanol .
    • Centrifuged for 5 minutes at 14000rpm.
    • Removed the supernatant and dried the pellet for 30- 45 minutes.
    • Dissolved the dried pellet in 30µl of TE buffer, then visualized the plasmid DNA under agarose gel electrophoresis.
    Agarose gel electrophoresis
    1. Prepared a 1% agarose gel by weighing 0.25g of the agarose into 25ml of TAE buffer( with 0.5µg/ml of ethidium bromide)
    2. Loaded 5µl of the plasmid DNA extracted into the wells on the agarose well.
    3. Ran the gel for 30minutes at 125 volts, 300mA.

    Restriction digestion
    Components
    Distilled water10µl
    Multicore buffer2µl
    E.coR11µl
    PST11µl
    Templates6µl
    Total20µl
    • A 20µl reaction mixture was prepared two sterile PCR tubes for each of the two different inserts, PETase and MHETase gene inserts.
    • The preparation was carried out on ice
    • Then incubated at 370C overnight followed by enzyme inactivation at 650C for 20 minutes, the reaction mixtures were stored at 40C .
    • Visualized the DNA after an agarose gel electrophoresis to determine the presence of inserts, as per their respective insert sizes, a 1kb DNA ladder was used to determine the insert sizes.
    Protein expression
    Materials
    • 100 ml LB broth with ampicillin (100ug/mL)
    • Overnight culture for the PETase and MHETase transformants.
    • Isopropyl β, D thiogalactoside (IPTG, 100µg/ml to 100ml of the LB broth).
    Procedure.
    • Inoculated 1ml of the overnight culture into 100ml of the LB- AMP broth
    • Incubated the prep at 37oC for 4 hours to acquire an OD>0.6
    • At an OD 0.6-0.8, picked off an aliquot of the cell suspension into a clean sterile eppendorf, kept on ice
    • Induced expression with 100µl of IPTG, for 4hours while picking 1 ml aliquots from the cell suspension after 1, 2, 3, 4 hours after induction, kept on ice.
    • Poured the rest of the cell suspension after the 4hours of protein expression into 50ml falcon tubes, centrifuged at >5000g, supernatant transferred to new falcon tubes, then proceeded for analysis of the cell pellet(cell lysis) and protein assays.
    • Prepared the collected 1ml aliquots for SDS PAGE to determine presence of the desired proteins