Materials

Procedure

1. Preheat water bath to 42º C.
2. Thaw competent cells on ice for 10 minutes.
3. Use 50 µL competent cells, add transforming DNA [up to 25 ng per 50 µL of cells, volume not exceeding 2.5 µL (5%)]. For control add 1 µL of control DNA (PUC19 carb resistance). Swirl to mix, store on ice 5 minutes.
4. Heat shock in 42º C water bath for 45 seconds.
5. Cool cells in ice bath for a few minutes.
6. Add 800 µL LB to each tube, set in shaker at 37º C for 45 minutes.
7. Transfer 200 µL of culture per plate (containing the appropriate antibiotic).
8. Spread using glass beads.
9. Invert plates and incubate overnight (12-16 hrs) at 37º C.

Procedure

1. Prepare 0.5 M PIPES (pH 6.7) (piperazine-1,2-bis[2-ethanesulfonic acid]) by dissolving 15.1 g of PIPES in 80 ml of pure H2O (Milli-Q, or equivalent). Adjust the pH of the solution to 6.7 with 5 M KOH, and then add pure H₂O to bring the final volume to 100 ml. Sterilize the solution by filtration through a disposable pre-rinsed Nalgene filter (0.45-µm pore size). Divide into aliquots and store frozen at -20°C. Prepare Inoue transformation buffer by dissolving all of the solutes listed below in 800 mL of pure H2O and then add 20 ml of 0.5 M PIPES (pH 6.7). Adjust the volume of the Inoue transformation buffer to 1 liter with pure H₂O.

	Reagent	Amount per liter	Final Concentration
	MnCl2•4H2O	10.88 g	55 mM
	CaCl2•2H2O	2.20 g	15 mM
	KCl	18.65 g	250 mM
	PIPES (0.5 M, pH 6.7)	20 ml	10 mM

Add H₂O to fill solution to 1 liter. Sterilize Inoue transformation buffer by filtration through a prerinsed 0.45-µm Nalgene filter. Divide into aliquots and store at -20°C.
2. Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for 16-20 hours at 37°C. Transfer the colony into 25 ml of SOB medium (LB may be used instead) in a 250-ml flask. Incubate the culture for 6-8 hours at 37°C with vigorous shaking (250-300 rpm).
3. At about 6 o'clock in the evening, use this starter culture to inoculate three 1-liter flasks, each containing 250 ml of SOB. The first flask receives 10 ml of starter culture, the second receives 4 ml, and the third receives 2 ml. Incubate all three flasks overnight at 18-22°C with moderate shaking.
4. The following morning, read the OD₆₀₀ of all three cultures. Continue to monitor the OD every 45 minutes.
5. When the OD₆₀₀ of one of the cultures reaches 0.55, transfer the culture vessel to an ice-water bath for 10 minutes. Discard the two other cultures.
6. Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for 10 minutes at 4°C.
7. Pour off the medium and store the open centrifuge bottle on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to walls of the centrifuge bottle or trapped in its neck.
8. Resuspend the cells gently in 80 ml of ice-cold Inoue transformation buffer.
9. Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for 10 minutes at 4°C.
10. Pour off the medium and store the open centrifuge tube on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to the walls of the centrifuge tube or trapped in its neck.
11. Resuspend the cells gently in 20 ml of ice-cold Inoue transformation buffer.
12. Add 1.5 ml of DMSO. Mix the bacterial suspension by swirling and then store it in ice for 10 minutes.
13. Working quickly, dispense aliquots of the suspensions into chilled, sterile microcentrifuge tubes. Immediately snap-freeze the competent cells by immersing the tightly closed tubes in a bath of liquid nitrogen. Store the tubes at -70°C until needed.

Materials

Procedure

Materials

Procedure

Procedure

1. Add 0.5 grams of agarose to 50 mL of 1X TAE buffer.
2. Microwave agarose/TAE until agarose completely dissolved.
3. Cool under water, add SYBR safe dye (2.5-3µL).
4. Pour into mold with appropriate comb.
5. Wait 15 minutes for gel to solidify.
6. Load DNA with dye into wells while submerged in 1X TAE.
7. Run gel at a constant 120V.
8. Check gel periodically.

Procedure

1. Prepare agarose gel and use 3 combs to make a bigger well.
2. Once it has run, use hand held UV lamp (in the dark, wearing goggles) to identify bands.
3. Cut out desired band with stamp pipette tip and transfer to a clean tube. The stamp pipette tip can be left in the tube to be cleaned out with a smaller pipette tip.
4. Weigh gel fragments and add 200 µL Buffer NTI for every 100 mg agarose gel.
Incubate sample for 5-10 min at 50º C, vortexing every 2-3 min until the gel is completely dissolved.
5. Load sample onto column over collection tube. Centrifuge for 30 sec at 11,000 x g.
6. Discard flow-through and replace column on tube.
7. Add 700 µL Buffer NT3 and centrifuge for 30 sec. at 11,000 x g. Discard flow-through.
8. Centrifuge for 2 min at 11,000 x g to completely remove buffer.
9. Transfer column to a 1.5 mL tube and add 20 µL ddH₂O.
10. Incubate at room temperature for 10 min.
11. Centrifuge for 1 min at 11,000 x g.

Materials

Materials

Procedure

1. Sediment the cells by centrifuging 1-5 mL of overnight LB-culture. Remove all medium.
2. Add 250 µL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is homogeneous.
3. Add 250 µL Lysis Buffer (L7). Mix gently by inverting the capped tube five time. Do not vortex. Incubate the tube at room temperature for 5 minutes.
4. Add 350 µL Precipitation Buffer (N4). Mix immediately, or for large pellets, vigorously shaking the tube until the mixture is homogeneous. Do not vortex. Centrifuge the lysate at >12,000 x g for 10 minutes.
5. Load the supernatant from the prior step on a spin column in a 2-mL wash tube. Centrifuge at the column for 12,000 x g for 1 minute. Discard the flow-through and place the column back into the wash tube.
6. Add 700 µL Wash Buffer (W9) with ethanol to the column. Centrifuge the column at 12,000 x g for 1 minute. Discard the flow-through and place the column into the wash tube. Centrifuge the column at 12,000 x g for 1 minute. Discard the wash tube with the flow-through.
7. Place the spin column in a clean 1.5 mL recovery tube. Add 30 µL of ddH2O to the center of the column. Incubate the column for 10 minutes at room temperature.
8. Centrifuge the column at 12,000 x g for 2 minutes. Discard the column. Store plasmid DNA at 4º C (short-term) or store the DNA in aliquots at -20º C (long-term).

Procedure

Procedure

Procedure

1. Log in to nanodrop program.
2. Moisten a Kim wipe and clean the pedestal.
3. Apply 2 µL H₂O to pedestal and click 'OK'.
4. Press 'Blank' button.
5. Wipe blank from pedestal using Kimwipe.
6. Apply 2 µL of desired sample to pedestal.
7. Click 'Measure'.
8. Print results.

Procedure

1. Grow cultures overnight in LB at 37 C, 150 RPM.
2. Measure OD₆₀₀ and dilute to get <0.01 OD₆₀₀.
3. Grow until the OD₆₀₀ approaches 0.5.
4. Load 96 well plate with LB, appropriate antibiotic, inducer stock solutions, and the appropriate volume of culture so as to reach an OD₆₀₀ of 0.1 in 200 µL.
5. Run Tecan program.

Procedure

1. Identify site that needs to be mutated.
2. Check the amino acid sequence to create a silent mutation, generally the last base in a codon.
3. Check a codon usage table to help choose how the codon should be changed, try to pick a frequently used codon.
4. Take about 20 base pairs upstream and 20 base pairs downstream of your desired mutation site to create your primer, try to have it start and end in a G or C. The sequence should be identical to the template except for the one changed base you are trying to mutate at the center.
5. The reverse primer will be the reverse complement of this sequence.

Materials

PCR program

1. 95º C 1 min
2. 95º C 50 sec
3. 60º C 50 sec Repeat Steps 2-4 17x (18x total)
4. 68º C 1 min / kb of plasmid
5. 68º C 7 min
6. 4º C Hold

Procedure

1. Thaw electrocompetent cells on ice, keep on ice when thawed.
2. Aliquot 20-30µL of competent cells into separate 1.5mL microcentrifuge tubes on ice.
3. Pipette appropriate amount of DNA (1 µL) into tube on ice.
4. Transfer cell/DNA into prechilled electroporation cuvette, keep on ice for 1 minute.
5. Make sure the electroporator is set to:
   Time constant = 4.5-5.0 ms
   Resistance = 200 W
   Capacitance = 25 mFD
   Volts = 1.7 kV (for 1mm gap cuvettes)
6. Electrocute cells once.
7. Add 1mL of LB to cuvette, pipette up and down to mix, and transfer mixture to 14mL Falcon culture tube.
8. Incubate for 1hr at 37C shaking at 150rpm.
9. Plate cells on appropriate antibiotic.