PROTOCOLS

gBlocks

1. Before opening the tube, spin it down in a microcentrifuge for 3–5 seconds to ensure the DNA is in the bottom of the tube.

2. Add IDTE to reach a final concentration of 50 ng/µL.

3. Vortex briefly.

4. Incubate at approximately 50°C for 15–20 minutes.

5. Ensure that the pellet of dried DNA is completely dissolved in IDTE

6. Briefly vortex and centrifuge

Resuspension of Primers

1. Find the oligo yield information (in nmol) on the tube label or specification sheet.

2. Multiply this number by 10.

3. The resulting product is the amount of buffer needed, in µL, to prepare a 100 µM solution.

4. Dilute the 100 µM stock to 10 µM as the working stock for all PCR reactions.

Q5^® High-Fidelity PCR

1. Transfer contents into a 0.2 mL PCR tube according to the following recipe:

Component	25µL Reaction	50µL Reaction (for gel extraction)
Q5 High-Fidelity 2× Master Mix	12.5 µL	25 µL
10 µM Forward Primer	1.25 µL	2.5 µL
10 µM Reverse Primer	1.25 µL	2.5 µL
Template DNA	20 ng	20 ng
Nuclease-Free Water	to 25 µL	to 50 µL

2. Set the PCR Thermocycling Conditions:

Step	Temperature	Time
Initial Denaturation	98°C	30 seconds
25-35 Cycles	98°C	10 seconds
	65°C	30 seconds
	72°C	30 seconds
Final Extension	72°C	2 minutes
Hold	4°C	-

Restriction Enzyme Digestion

1. Transfer contents into a 0.2mL PCR tube according to the following recipe:

Component	50µL Reaction
DNA	1µg
10X CutSmart Buffer	5µL (1X)
NheI-HF	1.0µL (20 units)
EcoRI-HF	1.0µL (20 units)
Nuclease-free Water	to 50µL

(2.) When further manipulation of DNA is required:

Heat inactivation (65°C for EcoRI-HF® & 80°C for NheI-HF®).
Remove enzyme by using Takara's Miniprep DNA Purification Kit # 9085.

Pre-heat the Elution Buffer or sterilized water to 60℃ can increase the elution efficiency.
For highest recovery, Incubate for 1 and a half hour / as long as possible at 37℃.

Remark: Incubate for 3 hours or overnight for better yield.

DNA Fragment purification

1. Add a 3X volume of Buffer DC to the PCR or other enzymatic reactions.

If the required volume of Buffer DC is less than 100 μL, add 100 μL of Buffer DC

Vortex briefly to mix the sample.

2. Place a Spin Column into a Collection Tube.

3. Pipette the sample from Step 1 into the Spin Column. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through.

Note: For improvement the recovery of DNA, transfer the flow-through to Spin Column and centrifuge again.

4. Add 700 μL Buffer WB to Spin Column. Centrifuge at 12,000 rpm for 30 seconds. Discard the flow-through.

Note: Make sure that the volume of 100% ethanol indicated on the bottle label has been added to the Buffer WB.

5. Repeat Step 4.

6. Place the Spin Column back into the Collection Tube. Centrifuge at 12,000 rpm for 1 minute.

7. Place the Spin Column into a new clean 1.5 ml tube. Add 25 −30 μL Elution Buffer or sterilized water to the centre of the membrane.

Let it stand for 1 minute at room temperature.

Note:

Pre-heat the Elution Buffer or sterilized water to 60℃ can increase the elution efficiency.
For highest recovery, Incubate for 1 and a half hour / as long as possible at 37℃.

Centrifuge at 12,000 rpm for 1 minute at room temperature to elute DNA.

Ligation

1. Transfer contents into a 1.5 mL microcentrifuge tube according to the following recipe on ice:

Component	20µL reaction
T4 DNA Ligase Buffer (10X)	2µL
Vector DNA	Varies
Insert DNA	Varies
T4 DNA Ligase	1µL
Nuclease-free water	to 20µL

2. Use NEBioCalculator to calculate molar ratios.

3. The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.

4. Gently mix the reaction by pipetting up and down and microfuge briefly.

5. Incubate at 16°C overnight or room temperature for 10 minutes.

Inoue transformation

1. Thaw the cells, then aliquot 100 μL of cells into a 1.5 ml microcentrifuge tube.

2. Add 10ng of DNA into corresponding tubes.

3. Incubates for 30 minutes on ice.

4. Heat shock at 42℃ for 45 seconds.

5. Incubates for 30 minutes on ice.

6. Add SOC medium so total volume is 1 mL.

7. Recover cells for 45 minutes in shaker. at 37°C at 270 r.m.p.

8. Centrifuge cells at 3000×xg for 3 minutes.

9. Remove all liquid medium from the tube.

10. Resuspend in 100 ul of LB medium.

11. Spread plates using glass plates.

Electrotransformation of E. coli

Preparation of electrocompetent cells

1. Inoculate a single colony of E. coli from a fresh agar plate into a flask containing 50 mL of LB medium. Incubate the culture overnight at 37 ̊C with vigorous aeration (250 rpm in a rotary shaker).

2. Inoculate 500 mL of prewarmed LB medium in a 2-L flask with 25 mL of the overnight bacterial culture. Incubate the flasks at 37 ̊C with agitation (300 cycles/min in a rotary shaker). Measure the OD600 of the growing bacterial cultures every 20 min. Begin to harvest the culture when the OD600 reaches 0.35.

3. As the OD600 of the cultures reaches 0.4, rapidly transfer the flask to an ice-water bath for 15–30 min. Swirl the culture occasionally to ensure that cooling occurs evenly. In preparation for the next step, place the centrifuge bottles in an ice-water bath. For maximum efficiency of transformation, it is crucial that the temperature of the bacteria not increase above 4 ̊C at any stage in the protocol.

4. Transfer the culture to ice-cold centrifuge bottles. Harvest the cells by centrifugation at 1000g for 15 min at 4 ̊C. Decant the supernatant and resuspend the cell pellet in 500 mL of ice-cold pure H2O by gentle up and down pipetting.

5. Harvest the cells by centrifugation at 1000g for 20 min at 4 ̊C. Decant the supernatant and resuspend the cell pellet in 250 mL of ice-cold 10% glycerol.

6. Harvest the cells by centrifugation at 1000g for 20 min at 4 ̊C. Decant the supernatant and resuspend the pellet in 10 mL of ice-cold 10% glycerol. Mix by gentle up and down pipetting.

Note: Take care when decanting because the bacterial pellets lose adherence in 10% glycerol.

(From Cold Spring Harb Protoc; doi:10.1101/pdb.prot101220 233)

Electropulsation

1. Mix 30-40 μL of cells with 1-5 μL of DNA in the electropulsation cuvette. Make sure there are no bubbles in the cuvette.

2. Apply pulse of 3-7 milliseconds at 6-9 kV/cm.

Expression of transformants

1. Immediately after the pulsation, add 1 mL of SOC medium. Shake the cuvette to mix.

2. Put the mixture on a sterile tube and incubate for 1h at 37°C. Shaking is optional.

3. Plate the cells on solid medium with ampicillin, usually adding 100μL of cells is sufficient.

Glass Bead Plating

1. Add 6 beads on either side of each plate.

2. Flicker tube until cells are uniformly distributed.

3. Gently pipette a dilution of outgrowth onto the centre of pre-warmed plate.

4. Place up to 4 plates on a folded paper towel square.

5. Vigorously move the plates forward and backwards. Make two complete 360° rotations, with stops every 90 degrees.

6. Allow plates to sit for 2 minutes.

7. Pour off the beads into a beaker.

8. Incubate plates at 37°C.

Transformation of UTEX2973 by Electroporation

1. Cells were grown to OD₇₅₀ of 0.5 (approx. 6 x 10⁸ cells/ml)

2. Wash once with a solution conting 2 mM Tricine, 2 mM EDTA (pH 8.0) of the same volume

3. Wash twice with double-distilled water

4. Resuspend in 10-fold concentration

5. Mix 40µl aliquot of the suspension with 4µg of plasmid DNA

6. Chill on ice for 2 minutes

7. Electroporated with a single square pulse with strength 8kV/cm^-1 for 5 ms

8. Immediately after pulsation, cells were transferred to fresh DTN medium to recover for 24 hours prior to antibiotic challenge in liquid culture

Transformation (E. coli)

1. Thaw competent cells on ice.

2. Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube.

3. Add 50 µl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4–5 times to mix the cells and DNA. Do not vortex.

4. Place the mixture on ice for 30 minutes. Do not mix.

5.Heat shock at 42°C for 30 seconds*. Do not mix.

Note: For the duration and temperature of the heat shock step as well as for the media to be used during the recovery period, please follow the recommendations provided by the competent cells’ manufacturer.

6. Add 950 µl of room temperature media* to the tube.

7. Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.

8. Warm selection plates to 37°C.

9. Spread 50–100 µl of the cells and ligation mixture onto the plates.

10. Incubate overnight at 37°C.

Plasmid Purification

1. Growth of bacterial cultures. Pick a single colony from a freshly streaked selective plate into 1 - 4 mL of liquid culture containing the appropriate selective antibiotic. Incubate at 37℃ overnight with vigorous shaking. (Growth for 12 - 16 hours and do not more than 16 hours, otherwise the bacterial cells will be hard to lyse and the yield of plasmid DNA will be reduced.)

Note: The culture volume should not be excessive because excessive bacteria will decrease lysis efficiency and result in poor purity of plasmid DNA.

2. Use 1 - 4 mL of the E. coli culture. Centrifuge at 12,000 rpm for 2 minutes to harvest the cell. Discard the supernatant.

3. Add 250 μL Solution l (containing RNase A). Resuspend the bacterial cell pellet completely by pipetting up and down.

Note: Be sure that the bacteria are completely resuspended, and no cell clumps remain before addition of Solution ll.

4. Add 250 μL Solution ll and mix gently by inverting the tube 5 - 6 times to completely lysis the cell until the solution becomes viscous and slightly clear.

Note: Do not allow the lysis reaction to proceed more than 5 minutes.

5. Add 350 μL of 4℃ precooling Solution lll and mix immediately and thoroughly by inverting the tube 5 - 6 times until a compact white pellet has been formed. Incubate at room temperature for 2 minutes.

6. Centrifuge at 13,200 rpm at room temperature for 10 minutes.

Note: Centrifuging at 4℃ is not recommended for precipitation.

7. Apply the supernatant from Step 6 into a new clean 1.5 mL tube by precise pipetting. Centrifuge at 13,200 rpm at room temperature for 10 minutes.

Note: The white precipitate will clog the spin column, reducing the yield.

8. Place a Spin Column in a Collection Tube.

9. Apply the supernatant from Step 6 onto the Spin Column by pipetting. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through with the aid of pipette.

10. Pipette 500 μL of Buffer WA onto the Spin Column. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through with the aid of pipette.

11. Pipette 700 μL of Buffer WB onto the Spin Column. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through with the aid of pipette.

Note: Make sure that the amount of 100% ethanol indicated on the bottle label has been added to Buffer WB.

12. Repeat Step 11.

13. Place the Spin Column back into new 1.5 mL tube without a lid. Centrifuge at 13,200 rpm for 5 minutes. Discard the flow-through with the aid of pipette.

14. Place the Spin Column back into 1.5 mL tube without a lid. Centrifuge at 13,200 rpm for 3 minutes. Discard the flow-through with the aid of pipette.

Note: Residual ethanol from Buffer WB may inhibit subsequent enzymatic reaction.

15. Place the Spin Column in a new clean 1.5 mL tube. Add 50 μL Elution Buffer or sterile purified water to the center of the Spin Column membrane. Incubate for 1 and a half hour / as long as possible at 37℃. Note: Pre-heat the Elution Buffer or sterile purified water to 60℃ will improve the elution efficiency.

16. Centrifuge at 13,200 rpm for 1 minute to elute DNA.

Colony PCR

1. Transfer contents into a 0.2 mL PCR tube according to the following recipe:

Component	25μL reaction	50μL reaction
10µM Forward Primer	0.5µL	1μL
10µM Reverse Primer	0.5µL	1μL
DNA	variable	variable
OneTaq 2X Master Mix with Standard Buffer	12.5μL	25μL
Nuclease-free water	to 25μL	to 50μL

2. Use a sterile toothpick to pick up individual colonies and dip into each reaction tube.

3. Create a stock of each individual colony by streaking the toothpick onto another agar plate containing the appropriate antibiotics and grow overnight.

4. Set the PCR Thermocycling Conditions:

Step	Temperature	Time
Initial Denaturation	94°C	2 minutes
30 Cycles	94°C	30 seconds
	54°C	60 seconds
	68°C	60 seconds
68°C	2 minutes
68°C	10°C	-

Gel Electrophoresis

1. Measure 0.2g of agarose.

2. Mix agarose powder with 20 mL 1xTAE in a microwavable flask.

3. Microwave until the agarose is completely dissolved.

4. Dilute the GelGreen® 10,000X stock reagent into the molten agarose gel solution at 1:10,000 and mix thoroughly. GelGreen® can be added while the solution is still hot.

5. Cast the gel and allow it to solidify. Load samples and run the gels (100V, 30 mins).

6. Image the stained gel with a 254 nm UV transilluminator or 490nm to 500nm visible light imaging system with a long path green filter.

Gel Extraction

The entire procedure should be executed with careful pipetting. Protocol in detail is as below.

1. Excise the agarose gel slice containing the DNA fragment with a clean, sharp scalpel under ultraviolet illumination. Cut as close to the DNA as possible to minimize the gel volume and increase the recovery rate of DNA.

Note: DNA should not be exposed to ultraviolet illumination for a long time in case of DNA damage.

2. Cut the gel into small pieces by cutting the gel. The gel melting time in Step 6 can be shortened and the recovery rate of DNA can be increased.

3. Add 600 μL Buffer GM to gel for melting.

4. Mix well and melt the gel at room temperature (15 - 25℃). Intermittent vortexing is essential to accelerate gel solubilization.

Note: Gel must be completely dissolved, or the DNA fragment recovery will be reduced. Extend the melting time when the gel concentration is high.

5. After the gel has been completely molten, check the color of the solution. If the color becomes orange or pink from yellow, add 10 μL of 3 M sodium acetate (pH5.2) to the solution and vortex well until the solution return to yellow. When the DNA fragment is smaller than 400 bp, add further with isopropanol to final concentration of 20%.

6. Set a Spin Column into Collection Tube.

7. Transfer the solubilized agarose from Step 7 into the column. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through. Transfer the flow-through to spin column again and centrifuge again.

8. Add 700 μL of Buffer WB into the Spin Column. Centrifuge at 12,000 rpm for 30 seconds. Discard the flow-through with the aid of pipette.

Note: Make sure that the amount of 100% ethanol specified on the bottle label has been added to the Buffer WB.

9. Repeat Step 8.

10. Place the Spin Column back into new 1.5mL tube without a lid. Centrifuge at 13,200 rpm for 5 minutes. Discard the flow-through with the aid of pipette.

11. Place the Spin Column back into 1.5mL tube without a lid. Centrifuge at 13,200 rpm for 3 minutes. Discard the flow-through with the aid of pipette.

12. Place the Spin Column into a new 1.5 mL tube. Add 30 μL of Elution Buffer or sterile distilled water to the centre of the membrane. Let it stand for 1 and a half hour / as long as possible at 37℃.

Note: Pre-heat the Elution Buffer or deionized water to 60℃ can improve elution efficiency.

14. Centrifuge at 13,200 rpm for 1 minute to elute the DNA.

Cre-lox Testing

1. Set up the reaction as follows:

panS DNA	X µL (389.4 ng)
10X Cre Recombinase Reaction Buffer	5 µL
Cre Recombinase	1 µL
H₂O	Up to 50 µL

2. Incubate at 37°C for 30 minutes and then 70°C for 10 minutes.

3. Perform gel electrophoresis.

Team:HK SSC/Experiments