Lysogeny Agar

Materials

• 10g of tryptone
• 5g of yeast extract
• 10g of NaCl
• 15g of agar
• 1L of Deionised Water

Procedure

1. Use a container with at least double the volume of the lysogeny agar that you are making.
2. Measure out the weights of tryptone, yeast extract, sodium chloride and agar with deionised water to 1L and mix well.
3. Ensure the lid is unscrewed by two and a half turns.
4. Send to be autoclaved.
5. Pour the plates next to a Bunsen burner.
6. Leave for 15-20 minutes to set/solidify.

Mini-Prep or Plasmid Purification

• Harvest bacterial cells
  1. Pellet 20ml of saturated E. coli for 60 seconds at 11,000 x g.
  2. Discard supernatant and remove as much liquid as possible.
• Lyse cells
  1. Add 500ml Resuspension Buffer P1 and resuspend cell pellet by vortexing.
  2. Split the solution into two 1.5ml microcentrifuge tubes.
  3. Add 250μl Lysis Buffer 2.
  4. Mix gently by inverting tube 8 times.
  5. Incubate at room temperature for five minutes or until lysate appears clear.
  6. Add 300μl Neutralization Buffer 3.
  7. Mix thoroughly by inverting tube 8 times.
• Clarification of lysate
  1. Centrifuge for 5 minutes at 11,000 x g at room temperature
  2. Put 500μl of Buffer PW1 per 1.5ml microcentrifuge tube used in heat block heated to 50^օC
• Bind DNA
  1. Place ISOLATE II Plasmid Mini Spin Column in a 2ml Collection Tube
  2. Pipette a maximum of 750μl of clarified sample supernatant onto column
  3. Incubate at room temperature for 2 minutes.
  4. Centrifuge for 1 minute at 11,000 x g and discard flow-through.
  5. Repeat stage 4 using the same ISOLATE II Plasmid Mini Spin Column and 2ml Collection Tube with the clarified sample supernatant from the other 1.5ml microcentrifuge tube from the same sample.
• Wash silica membrane
  1. Add 500μl Wash Buffer Pw1
  2. Centrifuge for 1 minute at 11,000 x g
  3. Add 600μl Wash Buffer PW2 (supplemented with ethanol)
  4. Centrifuge for 1 minute at 11,000 x g
  5. Discard flow-through and reuse Collection Tube
• Dry silica membrane
  1. Centrifuge for 2 minutes at 11,000 x g, to remove residual ethanol
  2. Place ISOLATE II Plasmid Mini Spin Column in a 1.5ml microcentrifuge tube.
• Elute DNA
  1. Add 50μl Elution Buffer P directly on the top of the silicon matrix
  2. Incubate at room temperature for 2 minutes
  3. Centrifuge for one minute at 11,000 x g.

Gel Electrophoresis

Materials

For a 1% Agarose Gel:

• 1g Agarose
• 100ml de-ionised water
• 10μl sybrsafe™
• Loading Buffer
• Masking Tape

Procedure

Make 1% Agarose Gel:

1. Dissolve 1g of agarose in 100ml of deionised water.
2. Microwave for 2 minutes and check it is all dissolved.
3. Wait for it to cool
4. Add the sybrsafe™ (10μl) pour the gel into the mold and leave it to set for 15 minutes.

Preparing DNA samples to load into wells in the gel.
Add loading buffer to your DNA samples to help visualise the DNA running through the gel.
Performing gel electrophoresis:

1. Inject your DNA samples into the appropriate wells and use a HyperLadder for reference (left hand side).
2. Turn on the machine and make sure the black lead is attached to the black end and the red lead is attached to the red end. Black is negative, Red is positive. The DNA will move towards the red because it is negative.
3. Leave gel running for at around 30 minutes.
4. Take to U:Genius Image Capture in biolab one to see the DNA bands under UV light. Do not leave the UV light on for too long before taking the photo as this can degrade the DNA.

Agarose Gel Extraction

1. Excise and dissolve gel slice
2. Using a clean scalpel excise DNA fragment from gel
3. Remove excess agarose, determine weight of gel slice and transfer into a clean tube
4. Add 200μl Binding Buffer CB per 100mg of 2% agarose gel
5. Incubate sample at 50^օC for ten minutes, vortexing sample briefly every 3 minutes until gel slice is completely dissolved
6. Incubate at room temperature for 2 minutes
Bind DNA
1. Place ISOLATE II PCR and Gel Column in a 2ml Collection Tube and load 600μl of the sample
2. Centrifuge for 30 seconds at 11,000 x g and discard flow-through
3. Reuse collection tube for step 3
Wash silica membrane
1. Add 700μl Wash Buffer CW to ISOLATE II PCR and Gel Column
2. Centrifuge for 30 seconds at 11,000 x g
3. Discard flow-through and place column back into collection tube
4. Repeat step three to minimize chaotropic salt carry-over
Dry silica membrane
1. Centrifuge for one minute at 11,000 x g, to remove residual ethanol
2. Place ISOLATE II PCR and Gel Column in a 1.5ml microcentrifuge tube

Elute DNA

1. Incubate at room temperature for 3 minutes
2. Add 15-30μl Elution Buffer C directly onto silica membrane
3. Incubate at room temperature for 3 minutes
4. Centrifuge for one minute at 11,000 x g.

SOC Media

Materials

To make 100ml SOC Media

• 2g Tryptone
• 0.5g Yeast Extract
• 2.5ml 400mM NaCl
• 625μl 400mM KCl
• 10ml 100mM MgCl₂
• 1ml 200mM Autoclaved and filter sterilised Glucose

Procedure

1. Weigh out tryptone and yeast extract into vessel suitable for autoclaving. Add the NaCl, KCl, MgCl₂ to the bottle.
2. Make up to 100ml with Distilled Water.
3. Make glucose solution in a vessel suitable for autoclaving.
4. Autoclave both solutions separately to avoid the reaction of glucose with other components.
5. Add 1ml glucose solution using a filter sterilisation syringe to the media.

Competent Cell Production

Materials

• 100ml LB + 5ml for overnight culture
• 100mM CaCl₂
• 85mM CaCl₂, 15% glycerol v/v

Procedure

1. Streak competent cells onto agar plate and incubate overnight at 37 ^oC
2. Prepare and autoclave above solutions.
   
3. Inoculate a single colony into 5ml LB in a 50 ml falcon tube. Grow overnight at 37 ^oC, shaking at 200rpm.
   
4. Keep solutions at 4 ^oC overnight, and LB at 37 ^oC so that when cells get transferred they do not experience a temperature change.
5. Pre-cool the rotor of the centrifuge.
   
6. Use 1 ml of overnight culture to inoculate 100ml of LB in a 250ml bottle. Shake at 37 ^oC for 1.5-3 hours, until OD650 reaches 0.4-0.6.
   
7. Put cells on ice for 10 mins (keep cold from now on, and cool everything on ice before adding). Split into 2 x 50ml falcon tubes.
   
8. Centrifuge in the big centrifuge for 3 mins at 5000 rpm.
   
9. Decant supernatant and gently resuspend in 5ml cold 100mM CaCl₂ by inverting tube slowly. (Cells susceptible to mechanical disruption)
   
10. Incubate on ice for 20mins
    
11. Centrifuge as before (3 mins at 5000 rpm)
    
12. Discard supernatant and resuspend in 2.5ml cold 100mM CaCl₂/ 15% glycerol v/v
    
13. Pipette into microtubes and freeze in -80^oC. (100µl per tube).

PCR

PCR with MyFi DNA Polymerase

• 5μl 5x MyFi Reaction Buffer
• 1ng Template
• 0.5μl of each 20μM Primer
• 1μl MiFi DNA Polymerase
• Water to 25μl

Colony PCR with MyTaq DNA Polymerase

Set up the reaction mixture as below:

• 10μl 5x MyTaq Reaction Buffer
• 1μl of each 20μM Primer
• 1μl MyTaq DNA Polymerase
• Water to 50μl
• Add at end: Template (cells)

Working under sterile conditions, use a pipette tip to touch the colony to be picked. Use this to mark a cross on a second, clean plate, then dip the tip into the PCR tube.

For colony PCR use 10 minute initial denaturing step to lyse the cells.

Thermal cycling conditions

Digestion

In a microcentrifuge tube, mix together:

• 500ng DNA
• 2 μl NEB buffer 2.1
• 1 μl Enzyme 1
• 1 μl Enzyme 2
• H2O to 20 μl

Flick tubes to mix and give a quick spin to collect at bottom of tube. Incubate at 37°C in heat block for 1hr.
Heat inactivate for 20mins at 80°C.

Ligation

This protocol uses NEB T4 DNA ligase

Use a molar ratio of 3:1 insert:vector DNA (or 3:3:1 if using 2 parts).

• 50ng vector DNA, corresponding moles of inserts
• 50 ng Vector
• H2O to 20 μl
• 2ul Ligase buffer
• 1ul T4 ligase

Incubate at room temperature for 1 hour.
Heat inactivate at 80°C for 20 minutes.

Transformation

If using DNA from the kit plates, resuspend DNA in the well in 10 μl water, pipetting up and down. Leave for 10 mins to fully resuspend.

1. Thaw tubes of 100μl competent cells on ice.
2. Add 1 μl DNA to each tube. (Use 1 μl for DNA from kit plates, or 4 μl from ligations).
3. Incubate cells on ice for 30 mins.
4. Heat shock for 60s in heatblock at 42°C.
5. Incubate on ice for 5 mins.
6. Add 500 μl SOC media to each tube. Incubate at 37°C for 2hrs, shaking (220rpm).
7. Plate 100μl on a plate with corresponding antibiotic resistance.