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PLASMID DNA ISOLATION - BY MINI PREP PROTOCOL

1. Clean the benches and pipettes.

2. Centrifuge 1mL of bacterial culture (in log phase of growth) for 7 minutes at 13000rpm in a microcentrifuge.

3. Pour off the supernatant, leaving abput 50 - 100uL.

4. Resuspend the bacterial pellet with 300uL of P1 buffer (50mM Tris-HCl, 10uM EDTA and 10ug/mL of RNAse)

5. Add 300uL of P2 buffer (0.2 M NaOH, 1% SDS) and mix by inversion.

6. Incubate the tubes for 5 minutes at room temperature.

7. Add 300uL of P3 buffer (3M potassium acetate solution (pH 5.5)), this solution neutralizes NaOH in the previous lysis step while precipitating the genomic DNA and SDS in a insoluble white, rubbery precipitate.

8. Mix by inversion.

9. Place on ice during 5 minutes.

10. Centrifuge for 10 minutes at 13000rpm.

11. Carefully, transfer the supernatant to a new microcentrifuge tube and discard the pellet.

12. Add 600uL of Phenol: chloroform: isoamyl alcohol (24:1:1).

13. Mix by inversion and centrifuge for 5 minutes at 13000rpm.

14. Transfer the aqueous phase (?700uL) to new properly labeled microtube.

15. Add an equal volume of cold isopropanol (?700uL) to the recovered supernatant. Mix by inverting the tube.

16. Pellet the DNA by centrifugation at 4°C for 15 minutes at 13000rpm.

17. Pour off isopropanol, being carefully not to loose pellet. Use a pipette tip to remove remaining isopropanol without dislodging the DNA pellet.

18. Rinse the pellet with 500uL of 70% ethanol. Mix by inversion and centrifuge briefly (5 minutes at 13000rpm).

19. Remove the residual ethanol and dry the DNA in the DNA SpeedVac.

20. Resuspend the DNA in 30uL of ultrapure water.

POLYMERASE CHAIN REACTION (PCR) - FOR AMPLIFICATION OF RH1AB GENE COMPLEX

1. Clean the bench and the pipettes.

2. Thawed the PCR mix, nuclease-free water, the primers, and the DNA samples. Place them on ice.

3. Mix the reagents in each PCR tube, in the following order: (If you planned to make a "Master Mix", calculate the volumes for the amount of samples plus 1 tube). Finally add 2uL of the DNA isolate.

Reagent Volume for 1 tube

Nuclease free water 5.5uL

Platinum® Blue PCR Super Mix 2X 12.5uL

rht 1bF forward primer 2.5uL

Rht 2bR Reverse primer 2.5uL

Premix volume (per tube) 23uL

Amount of DNA sample (per tube) 2uL

4. Turn the thermal cycler on and set the Protocol: rh1AB.amp (within iGEM user).

5. Put the PCR tubes on the thermal cycler. Insert volume data and begin the program.

6. When the equipment reaches the last temperature setting (at 4°C), take off the tubes and turn off the equipment.

7. Use PCR products immediately. Otherwise, keep them at 4°C if they will be used within the first 24 hours or at -20°C to keep for longer periods.

AGAROSE GEL ELECTROPHORESIS

1. Assemble the gel casting tray and comb. The comb should not touch the bottom of the tray.

2. Add 0.7g of agarose to 70 mL of 1X TAE Buffer. Using a microwave, melt the agarose solution (for 1 minute).

3. When the agarose solution has cooled (to about 50°C), add 2uL of ethidium bromide solution and mix it well.

4. Pour solution directly into the casting tray, ensuring that no bubbles get into the gel. (rinse the flask immediately).

5. Allow the gel to cool. It will solidify and become slightly opaque within 20 to 30 minutes. Remove the gel casting and set in the correct position.

6. Submerge the gel by adding approximately 800mL of 1X TAE running buffer to cover the gel by about a half a centimeter.

7. Carefully remove the comb by lifting it gently at one end, tilting the comb as it comes out. Pulling the comb straight up creates a vacuum in the wells, which tends to lift the whole gel out of the tray. Ensure that the wells are submerged and filled with buffer.

8. Prepare the 2uL DNA ladder (marker) for loading using 2uL of loading buffer.

9. Load a maximum of 10uL of each sample into individual wells with a gel loading tip.

10. Once all the samples are loaded place the cover on the gel apparatus. Connect the leads so that the red (positive) lead is at the end of the gel to wich DNA will migrate and the black (negative) lead is is at the side of the gel containing the wells. Turn on the power supply and set at 100V. Check the gel after minutes.

11. When the blue tracking dye (which runs in these gels along with a DNA fragment of about 200-400bp) has migrated about 50% of the distance to the end of the gel (usually within 60 minutes), turn off the power supply and disconnect the power leads.

12. Transfer the gel to the transilluminator and visualize DNA with UV light. The DNA fragment band corresponding rhlAB gene complex has 2343bp.

TRANSFORMATION

1. Thaw the competent cells on ice for 5 minutes (competent cells are extremely susceptible to heat, work with them always on ice.

2. Meanwhile the cells are thawing out, add 2-4uL of DNA to a 2 mL microtube on ice (use molecular grade H2O for your negative control).

3. Add 50uL of the thawed out competent cells to 2mL microtube.

4. Incubate 5 minutes on ice.

5. Add 200uL of room temperature SOC medium to the microtubes. (It is no longer necessary to keep the cells on ice from this step onwards).

6. Incubate for 1 - 2 hours at 37°C with vigorous shaking.

7. Spread 100uL to the transformants onto an agar plate with the appropriate antibiotic.

8. Incubate at 37°C "overnight" with the top side looking downwards

AMPICILLIN STOCK

1. Weight the appropriate amount of antibiotic powder for the amount of H20dd to be used. (we use 0.25g in 5mL of H20 to make a 50mg/ml solution).

2. Absorb your solution with the sterile syringe.

3. Filter your solution into the sterile 15mL centrifuge tube.

PREPARING CHEMICALLY COMPETENT CELLS

1. Grow an overnight cell culture to the strain you want to turn competent.

2. Make a 1:100 dilution of the overnight cell culture and grow to a OD600 of 0.3 - 0.5 (we inoculate 1mL of overnight into 100 mL of LB)

3. Split the cell culture into two 50 ml centrifuge tubes.

4. Centrifuge at 4000rpm at 4°C for 10 minutes (Always keep the cells on ince from this point onward, this is very important).

5. Discard the supernatant.

6. Gently resuspend the cells using 20mL of cold 0.1M CaCl2 solution for each centrifuge tube.

7. Incubate on ice for 30 minutes.

8. Centrifuge at 4000rpm at 4°C for 10 minutes

9. Discard supernatant.

10. Resuspend each tube using 3mL of cold CaCl2 0.1M 15% glycerol solution.

11. Aliquot however you seem fit in 2mL microtubes or cryovials.

12. Store at -80°C as soon as you are done.

AGAR PLATES

1. Mix 20g of agar powder for every 1000mL of LB Broth being used.

2. Autoclave

3. Wait till the temperature has gone down to a point where it won't burn to touch and add the appropriate antibiotic at the desired concentration.

4. Serve 25ml of agar + LB Solution per petri dish.

GLYCEROL STOCK

1. Grow an overnight cell culture of the desired sample.

2. Add 1ml of overnight culture into a 2mL microtube or cryovial.

3. Add 1mL of 40% glycerol solution into the same tube.

4. Store at -80°C (To reactivate simple inoculate 50uL into fresh LB Broth with the appropriate antibiotic).

RESTRICTION DIGEST

1. Quickly vortex all ingredients (Buffer, BSA, DNA from Miniprep) before beginning

2. Add the following in a microcentrifuge tube:

1. 5uL of Buffer (NEBuffer2).

2. 1uL of BSA.

3. 0.5 picomoles DNA, normally uses 10uL of miniprep or 5uL of purified PCR product.

4. Water to make 48uL

3. Vortex enzymes and add 1uL of each to the tube.

1. If you are digesting purified PCR products, add 1uL of Dpnl to the reaction.

4. Incubate reaction a 37°C water bath for at least one hour.

1. If your digesting a "vector", add 1uL Antarctic Phosphatase and 6uL of Phosphatase buffer after 2 hour of incubation and incubate for another hour.

2. See Bio-Brick Assembly Schedule for more details.

3. 5 Heat kill the digest for 20 minutes at 80°C.

4. Sore digested DNA in the refrigerator (4°C) for use in the very near future.

LIGATION

1. Mix together the following:

a. 11uL of deionized water.

b. 2uL of Reaction Buffer for T4 Ligase.

c. 2uL digested "Vector" (Phosphatased).

d. 4uL digested "Insert" (PCR product digested with Dpnl).

e. 1uL of DNA ligase.

2. Incubate at room temperature for 30 minutes (or 4°C overnight for blunt ends).

3. Kill Reaction for 15 minutes at 65°C.

Notes: If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.

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