Protocols

LB Media

950 mL dH₂0

10 g Tryptone

10 g NaCl

5 g Yeast Extract

1 L Total

Add 15 g Agar, if being poured into plates.

Autoclave, when cool add antibiotics if desired.

Antibiotic Stock Solutions

Chloramphenicol

Working Concentration 12.5 μg/ml

Stock solutions can be made at 35 mg/ml in ethanol and kept at -20º C

Kanamycin

Filter sterilize for kanamycin

Working Concentration:

25 μg/mL for low-copy plasmids

35 µg/mL for high-copy plasmids

Stock solution is 35 mg/ml in water and kept at -20º C

Spectinomycin

Filter sterilize

Working Concentration 50 μg/mL

Stock solution is 100 mg/mL in water and kept at -20º C

Heat Shock Transformation

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.

Making Chemically Competent Cells

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.

Double Restriction (Fast) Digest

~20 µL DNA (as much as possible)

1 µL Restriction Enzyme 1

1 µL Restriction Enzyme 2

5 µL 10X Universal Buffer (with loading dye)

Add appropriate amount of dH₂0 (µL)

50 µL Total → 37º C, 3 hrs.

Treat insert with XbaI and PstI

Treat vector with SpeI and PstI

Ligations (Standard Assembly)

Use Excel macro to determine vector/insert volumes based off the DNA concentrations, where a vector to insert ratio is maintained at 3:1 and the desired vector mass in the reaction 200 ng.

Ligation reaction:

__ µL vector DNA

__ µL insert DNA

2 µL T4 10x Buffer

1 µL DNA ligase

Add appropriate amount of dH₂0 (µL)

20 µL Total →Leave at room temperature for 20 minutes

Vector control:

__ µL vector DNA (same volume as ligation reaction)

2 µL Buffer

1 µL ligase

Add appropriate amount of dH₂0 (µL)

20 µL total

Insert control:

__ µL insert DNA (same volume as ligation reaction)

2 µL Buffer

1 µL ligase

Add appropriate amount of dH₂0 (µL)

20 uL Total

Gel Electrophoresis

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.

Gel Extraction and Purification

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.

PCR Amplification for Golden Gate Assembly

10 µL 5x HF Buffer

1 uL dNTPs

2.5 µL Forward Primer

2.5 µL Reverse Primer

100 ng Template DNA (2 ng/µL)

0.5 µL DNA Phusion Polymerase

Add appropriate amount of ddH₂O

50 µL Total

Run 1% agarose gel for verification. If the gel is good, perform PCR clean up.

Golden Gate Assembly

100 ng for each DNA part

1 µL BsaI

1 µL T4-ligase

2 µL 10X T4 ligase buffer

Add appropriate amount of ddH₂0.

20 µL Total

DNA Extraction (Minipreps)

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).

Making and Reviving Glycerol Stocks

Add equal volumes (500-700 µL) of overnight cell culture and glycerol into a cryotube, keep sterile with a flame.

Store at -80º C.

When reviving a glycerol stock, keep the glycerol stock on dry ice.

Use a pipette tip to poke and/or slightly swirl glycerol stock and drop tip into 5 mL LB culture with appropriate antibiotic and shake overnight.

Sequencing Prep

Primer will have [ng] content printed on label: add 10x H₂0 for DNA at 100 uM.

Need 10 uM for sequencing, so dilute a portion of the hydrated primer solution 10x.

Determine DNA concentration of template DNA.

(Premixed) in 0.5 µL tube

.6 µg DNA (final concentration: 50 ng/µL)

8 pmol primer (universal primers 10 µM = .8 µL)

Add appropriate amount of H₂O.

12 µL total

Measuring DNA Concentration

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.

Tecan Testing Protocol

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.

Primer Design for Site-Directed Mutagenesis PCR

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.

Site-Directed Mutagenesis PCR

Primer will have [µg] content printed on label: add H₂0 1:1 for DNA at 1 µg/µL.

Need 0.1 µg/µL for PCR reaction, so dilute a portion of the hydrated primer solution 10x.

Determine DNA concentration of template DNA.

50 ng Template DNA

5 µL 10x Turbo Buffer

1 µL Forward Primer (0.1 ug/uL)

1 µL Reverse Primer (0.1 ug/uL)

1 µL dNTPs (10 mM)

1 µL Pfu Turbo (enzyme)

Add appropriate amount of dH₂O.

50 µL Total

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

Electroporation Transformation