Plasmid resuspension Protocol

Requirements:

• 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.

• LB broth (100ml)
• LB agar
• Competent cells, E.coli BL21
• Resuspended plasmid DNA (300ng/µl)
• Ampicillin (100µg/ml)
• Ice, incubator, centrifuge.

• 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.

• 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.

• 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.

• 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 -80^oC 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.

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.

• 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.

• 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).

• Inoculated 1ml of the overnight culture into 100ml of the LB- AMP broth
• Incubated the prep at 37^oC 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

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