950 mL dH20 10 g Tryptone 10 g NaCl 5 g Yeast Extract 1 L Total (add dH20 to reach total volume) Add 15 g Agar, if being poured into plates. Autoclave, when cool add antibiotics if desired.

Materials

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

Procedure

1. Preheat water bath to 42º C.
2. Thaw competent cells on ice for 10 minutes.
3. Use 50 µL chemically 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 (e.g. PUC19). Swirl gently 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 1-2 minutes.
6. Add 800 µL LB to each tube, set in shaker at 37º C for 45 minutes.
7. Transfer 200 µL of culture to a 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

~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 → incubate at 37º C for 3 hrs.

Procedure

Treat insert with XbaI and PstI

Treat vector with SpeI and PstI

After incubating at 37º C for 3 hrs, either heat inactivate the enzymes or proceed directly to gel extraction and purification.

Materials

Ligation reaction:

__ µL vector DNA

__ µL insert DNA

2 µL T4 10x Buffer

1 µL DNA ligase

Add appropriate amount of dH₂0 (µL) to reach total volume

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) to reach total volume

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) to reach total volume

20 uL Total

Procedure

Mix these materials in the amounts determined by the reaction volume calculator for the vector and insert DNA here.

Procedure Add 0.5 grams of agarose to 50 mL of 1X TAE buffer. (1% agarose gel) Microwave agarose/TAE until agarose is completely dissolved. (Caution: Will be hot) Cool under water or let sit until cool. Add SYBR safe dye (2.5-3µL). Pour into mold with appropriate comb. Wait 15 minutes for gel to solidify. Transfer gel to a gel electrophoresis box. Load DNA and appropriate DNA standard or ladder with dye into wells while submerged in 1X TAE buffer. Run gel at a constant 120V. Check gel periodically until the dye has run the desired length of the gel. View gel under short wavelength or UV light, with proper protection, to check for bands.

Procedure

1. Prepare agarose gel (see gel electrophoresis) and use additional or larger combs to make larger wells.
2. Once the gel has run, use preferably a blue light lamp, or UV for a very short time (it will degrade your DNA), (in the dark, wearing goggles and skin protection) to identify bands.
3. Cut out the desired bands with a stamp pipette tip or clean razor 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.

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. Load sample onto column over collection tube. Centrifuge for 30 sec at 11,000 x g. Discard flow-through and replace column on tube. Add 700 µL Buffer NT3 and centrifuge for 30 sec. at 11,000 x g. Discard flow-through. Centrifuge for 2 min at 11,000 x g to completely remove buffer. Transfer column to a 1.5 mL tube and add 20 µL ddH2O. Incubate at room temperature for 10 min. Centrifuge for 1 min at 11,000 x g.

Materials

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 to reach total volume

50 µL Total

or for DNA with high G/C content we found a combination of Taq and Pfu gave us better results.

5 µL 10x Buffer

10 uL Q solution

2.5 µL Forward Primer

2.5 µL Reverse Primer

1.25 µL DNTPs

100 ng Template DNA (2 ng/µL)

0.3 µL Taq DNA Polymerase

0.1 µL Cloned Pfu DNA Polymerase

Add appropriate amount of ddH₂O to reach total volume

50 µL Total

PCR program

1. 98º C 30 sec
2. 98º C 10 sec
3. ___º C 30 sec (Temperature depends on Tm of your primers)
4. 72º C 1 min / kb Repeat Steps 2-4 29x (30x total)
5. 72º C 5 min
6. 4º C Hold

Run PCR products on a 1% agarose gel for verification. If the gel is good, perform PCR clean up with your kit of choice.

Materials

40 fmol of DNA for each part (or 100 ng if your parts are all roughly the same size)

.75 µL BsaI

.2 µL 100x BSA

1 µL T4 DNA ligase

2 µL 10X T4 ligase buffer

Add appropriate amount of ddH₂0 to reach total volume.

20 µL Total

To convert your DNA concentration to fmol/µL, use the equation 1µg of 1kb DNA = 1.52 pmol.

PCR Program

1. 37º C 2 min
2. 16º C 3 min Repeat Steps 1-2 49x (50x total)
3. 50º C 5 min
4. 80º C 5 min
5. 4º C Hold

Following PCR, directly transform 5-10 µL of your product into competent cells.

Procedure (Invitrogen Quick Plasmid Miniprep Kit)

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

Add equal volumes (500-700 µL) of overnight cell culture and 80% 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.

Procedure (Quintara Biosciences)

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

Need 10 µM solution for sequencing, so dilute a portion of the hydrated primer solution 10x (e.g. 1 µL primer + 9 µL H₂0).

Determine DNA concentration of template DNA.

(Premixed) in 0.5 µL tube

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

8 pmol primer (10 µM = .8 µL)

Add appropriate amount of H₂O for total volume.

12 µL total

Procedure Log in to nanodrop program and select Nucleic Acids/DNA. Moisten a Kimwipe and clean the pedestal. Apply 2 µL H2O to pedestal and click 'OK'. Press 'Blank' button. Gently wipe blank from pedestal using a Kimwipe. Apply 2 µL of desired sample to pedestal. Click 'Measure'. Gently wipe pedestal using a Kimwipe and continue measuring remaining samples. Record/print results.

Procedure

1. Grow cultures overnight, or until OD₆₀₀>1, 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, or other media of choice, depending on the experiment, as well as the appropriate antibiotic, inducer stock solutions, and the appropriate volume of culture so as to reach an OD₆₀₀ of 0.1 in 200 µL. If changing media, first spin down the culture to pellet, decant and aspirate any remaining media, and resuspend in new media to reach the desired OD₆₀₀ before loading in plate.
5. Be sure to include the appropriate positive and negative controls (e.g. blank media, strain control, etc.).

Tecan Program Parameters Temperature: 37 C Orbital Shake Frequency: 244.5 rpm (Amplitude of 2.5 mm) Orbital Shake Duration: 600 sec Excitation wavelength: 485 nm Emission wavelength: 535 nm Absorbance wavelength (for OD readings): 595 nm Gain: 35

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 for your chassis 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. 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

Primer will have [µg] content printed on label: add dH₂0 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 (e.g. 1 µL primer + 9 µL dH₂0).

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 to reach total volume.

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

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.

Materials

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.

Amplification

100 ng Template DNA

10 µL HF 5x Buffer

2.5 µL Forward Primer (0.1 µg/µL)

2.5 µL Reverse Primer (0.1 µg/µL)

1 µL dNTPs (10 mM)

0.5 µL Phusion Polymerase (enzyme)

Add appropriate amount of dH₂O.

50 µL Total

PCR program

1. 98º C 30 sec
2. 98º C 10 sec
3. 55º C 30 sec
4. 72º C 1 min / kb Repeat Steps 2-4 29x (30x total)
5. 72º C 5 min
6. 4º C Hold

Purify the PCR product and determine the resultant concentration

SOE PCR

300 ng Template DNA (larger part)

xx ng Template DNA (smaller part) keeping a 1:1 molar ratio

10 µL HF 5X Buffer

2.5 µL Forward Primer (0.1 µg/µL)

2.5 µL Reverse Primer (0.1 µg/µL)

1 µL dNTPs (10 mM)

0.5 µL Phusion Polymerase (enzyme)

Add appropriate amount of dH₂O.

50 µL Total

PCR program

Same as above.

When to Use

Transformation of competent cells may yield false positives. Through colony PCR, one may screen for the presence of the assembled sequence by amplifying with the universal forward and reverse primers and checking the length of the fragments through gel electrophoresis.

Materials

2 µL 10x Buffer

4 µL Q solution

1 µL Universal Forward Primer (0.1 µg/µL)

1 µL Universal Reverse Primer (0.1 µg/µL)

0.5 µL dNTPs (10 mM)

1.375 µL dH₂O

Pick a colony with a sterile tip and set the tip in 10 µL dH₂O in a PCR tube for 10 minutes. Pipet up and down to resuspend the cell material and then add the reagent, DNA, and buffer mixture. While picking the colony, some cells should also be transferred to a new plate with the same antibiotic so that if the screen results are positive, the colony may be identified and grown up.

20 µL Total

PCR program

1. 98º C 30 sec
2. 98º C 10 sec
3. 55º C 30 sec
4. 72º C 1 min / kb Repeat Steps 2-4 29x (30x total)
5. 72º C 5 min
6. 4º C Hold

Materials

• 1x M9 salts
• 2 mM MgSO₄
• 0.1 mM CaCl₂
• 0.4% carbon source (eg. glucose)
• Dissolve in sterile dH₂0