Polymerase Chain Reaction

Phusion HF Polymerase Protocol

Per tube, add in this order:

PCR Reaction in Thermal Cycler

Colony PCR

1. Prepare 10 µl of cloning water per colony to be picked in PCR tubes.

2. Prepare culture tube filled with 4mL LB, 4µL resistance

3. Pick colony and resuspend in cloning water by pipetting up and down

4. Next, drop the tip inside the culture tube, or dot onto pre-numbered LB plate

5. Heat water with colonies for 10 minutes in 98°C; use as “template DNA”

6. Put culture tubes in 37°C shaker to incubate (overnight) while PCR is running.

7. Go TAQ PCR mix

8. TAQ PCR Protocol

9. Run Gel Visualization

10. Add 15µl PCR product (no need 6x loading dye)

11. Freeze & Miniprep cultures that have desired bands

Gradient PCR

Annealing Temperature: 5 °C below to 5 °C above.

Example: 62°C = 57°C -> 67°C

Increase Annealing Time to 30 seconds

Making a Gel for Gel Electrophoresis

1. 0.7g of Agarose

2. Add 100 ml 1x TAE buffer into an Erlenmeyer flask

3. Microwave for 2 minutes

4. Cool down under tap water for 20 seconds

5. Add 5 µl Sybrsafe

6. Pour the gel into mold with comb. Let cool for 30 minutes.

7. Remove the comb.

8. Transfer tray into an electrophoresis chamber, wells facing black side (anode) of chamber

9. Fill chamber with 1x TAE until TAE just covers the gel

Gel visualization/extraction

1. Prepare samples

2. Add DNA ladder Standard into first well.

3. Pipet prepared samples into rest of wells.

4. Seal electrophoresis chamber with chamber cap and plug cables in VWR power unit.

5. Set 125V, Run for 30 minutes (or longer) depending on length of sample expected.

6. Remove Gel tray once finished and visualize using trans-illuminator.

7. Extract if necessary into a preweighed 1.5 ml microcentrifuge tube.

Gel purification

Using Zymoclean Gel DNA Recovery Kit

1. Add 3 volumes (µl) for each volume of agarose (mg) excised from the gel.

2. Incubate at 55°C for 10 minutes until gel is dissolved

3. Pipette into a Zymo-Spin Column & Collection Tube

4. Centrifuge (at 16000 x g)for 1 minute, and discard the flow-through.

5. Add 200µl DNA wash Buffer and centrifuge for 30 seconds.

6. Add another 200µl DNA wash buffer and centrifuge for a minute.

7. Place the spin column into a new sterile microcentrifuge tube.

8. Add up to 30µl of Cloning Water directly to the column matrix.

9. Let it sit for 4 minutes, and centrifuge for 4 minutes.

Treating with DPNI

1. Add to a PCR rxn mix after PCR.

2. 1µl DPNI per 50 µl rxn mix.

3. Let it run 37°C for 1 hour.

DNA Purification

Using Zymoclean DNA Clean & Concentrator

1. Add 5 volumes of DNA binding Buffer to PCR product or Short DNA fragments.

2. Vortex Tube.

3. Load mixture into Zymo-Spin Column and Collection Tube

4. Centrifuge (16000 x g) for 30 seconds. Discard flow-through.

5. Add 200µl of DNA Wash Buffer and centrifuge for 30 seconds.

6. Add another 200µl of DNA Wash Buffer and centrifuge for 2 minutes.

7. Place the spin column into a new sterile microcentrifuge tube.

8. Add up to 30µl of Cloning Water directly to the column matrix.

9. Let it sit for 4 minutes, and centrifuge for 4 minutes.

Golden Gate Digestion/Ligation

1. Design Primers accordingly, taking into account Type IIs Restriction Enzyme site.

2. Add purified pieces you want to ligate based on concentration results (100ng / x ng/µl) into a PCR tube.

3. Add cloning water to the mix up to 10.5µl total.

4. Add 1.5µl Cutsmart Buffer to the mix.

5. Vortex ligase buffer. Add 1.0µl to the mix.

6. Add 1µl of Type IIs restriction enzyme (we usually use sapI)

7. Add 1µl of T4 Ligase to the mix.

8. Total should be 15µl in the tube. If volume of pieces you want to ligate together add up to greater than 10.5µl, scale up the mix proportionally.

Reaction assembly

Restriction Enzyme Digestion

1. Follow NEB protocols.

2. Find appropriate buffer if doing double digests.

3. Heat inactivate at the end.

4. If doing sequential digests, heat inactivate in between each step.

Blunt End Ligation

1. Add 18µl of purified PCR product into a PCR tube.

2. Add 2.25µl of 10x T4 Ligase Buffer

3. Add 1.125µl of T4 PolyNucleotide Kinase.

4. Put in thermocycler for 37°C for 45 minutes.

5. Add 1.125µl T4 DNA Ligase

6. Leave at room temperature for at least 1 hour.

7. Heat inactivate whatever you are not using at 65°C for 20 minutes.

Transformation (Electroporation)

1. Put electroporation cuvette on ice and thaw electro-competent cells for 5 minutes.

2. Prepare 500µl LB and a labeled culture tube.

3. Add up to 5µl of DNA directly to the competent cells. (usually use 2µl)

4. Transfer mix into cuvette, ensuring no bubbles and mix touching both sides.

5. Wipe down cuvette to remove moisture.

6. Place in electroporation machine and start.

7. If arc occurs, try with smaller amounts of DNA.

8. If successful, immediately add the LB directly into the cuvette.

9. Transfer LB + transformed cells into the culture tube.

10. Record time and incubate in the 37°C shaker for 1 hour.

Plating

1. Prepare two appropriate antibiotic resistance “100µl” and “Rest”

2. Add 100µl of transformed culture (after the 1 hour incubation) onto the 100µl plate.

3. Use a sterile spreader to streak the liquid culture onto the plate in the order on the left:

4. On the “Rest” Plate, pour the rest of the liquid culture and spread evenly on the plate.

5. Flip and put in the 37°C incubator overnight (no longer than 20 hours).

6. Wrap your plate after sufficiently grown and store in the 4°C fridge for up to 45 days.

Liquid Cultures

1. Determine volume of culture to grow in a culture tube. We usually use 4ml of LB.

2. In the biosafety hood, add 4ml of LB, and 4µl of each appropriate resistance(s).

3. Use a tip to pick up a single colony from the plate.

4. Drop the tip into the media.

5. Cap the culture tube to the first stop.

6. Incubate in 37°C shaker at 250rpm for up to 16 hours.

Freeze Cultures

1. Add 500µl of 30% glycerol into a cryo-freeze tube.

2. Add 500µl of overnight culture

3. Store in -80°C

Plasmid Miniprep

Using Zyppy Plasmid Miniprep Kit

1.Centrifuge overnight culture for 10 minutes at 5000 x g

2. Discard supernatant. Resuspend with 550µl LB

3. Transfer culture to 1.5 ml microcentrifuge tube.

4. Add 100 µl of 7X Lysis Buffer (Blue). Shake well.

5. Within 2 minutes, add 350 µl of cold Neutralization Buffer (Yellow). Shake well.

6. Centrifuge at 16000 x g for 4 minutes.

7. Transfer supernatant into a Zymo-Spin Column and Collection Tube.

8. Centrifuge at 16000 x g for 30 seconds. Discard flow-through.

9. Add 200µl Endo-Wash Buffer. Centrifuge at 16000 x g for 30 seconds.

10. Add 400µl Zyppy Wash Buffer. Centrifuge at 16000 x g for 2 minutes.

11. Transfer column into new labeled microcentrifuge tube.

12. Add 30µl of cloning water directly to column matrix. Let sit for 4 minutes.

13. Centrifuge at 16000 x g for 4 minutes to elute.