Biology Protocols

For 1litre: 

Measure ~900ml of distilled H₂O.

Add 16g Bacto Tryptone.

Add 10g Bacto Yeast Extract.

Add 5g NaCl.

Adjust pH to 7.2 with 5M NaOH.

Adjust to 1L with dH₂O.

Sterilize by autoclaving.

For 1litre: 

Measure ~900ml of distilled H₂O.

Add 10g Bacto Tryptone.

Add 5g Bacto Yeast Extract.

Add 5g NaCl.

Add 15g of Bacto Agar

Adjust pH to 7.2 with 5M NaOH.

Adjust to 1L with dH₂O.

Sterilize by autoclaving.

For 1litre: 

Measure ~900ml of distilled H₂O.

Add 10g Bacto Tryptone.

Add 5g Bacto Yeast Extract.

Add 5g NaCl.

Adjust pH to 7.2 with 5M NaOH.

Adjust to 1L with dH₂O.

Sterilize by autoclaving.

• Grow up the cells in 100ml volumes of LB.
• Spin down the cells in 250ml buckets at 5K for 15 min in an RC-5B or equivalent.
• Pour off the supernatant and carefully resuspend the cells in 6.6ml of 25% sucrose. Avoid any pellet by repeatedly pipetting.
• Working on ice, add 1.34ml of fresh lysozyme solution (5mg/ml in 25% sucrose).
• Leave on ice for 5 min.
• Add 2.6ml of 0.25M EDTA.
• Leave on ice for 5 min.
• After 5 min remove a 1ml sample, add 1ml of Triton solution (1% Triton X-100 in 0.1MTris.HCl pH8) and gently mix. If the cells lyse - indicated by the solution becoming thick and viscous, proceed to the next step. If this does not occur leave the cells for another 5 min.
  (If the cells are very hard to lyse it may be worth carrying out the lysis at R/T or maybe adding Proteinase K).
• Add 10.5ml of the Triton solution to each of the complete buckets carefully down the side of the bucket so that the Triton floats on the surface.
• NB Add 1ml less to the bucket from which the sample was removed.
• Carefully mix the two layers by repeatedly pipetting through an inverted 10ml pipette until lysis occurs.
• Centrifuge at 10k for 30 min in a Sorval RC-5B or equivalent.
• Carefully remove the cleared lysate from the viscous pellet - it is a bit like separating eggs.
• Add a 1/2 volume of 30% PEG solution. Leave at room temperature for 30 min.
• Spin down the precipitated nucleic acids (etc) in a 50ml centrifuge tube at 10K for 10 min in a Sorval RC-5B.
• Resuspend the pellet in 11ml of TE buffer - you may need a whirlimixer.
• Add 11.38g of CsCl, 0.22ml TE and 0.35ml of ethidium bromide. Fill a quick seal centrifuge tube and centrifuge in the L8 for 16-20 hr.
• Remove the band containing the plasmid DNA (normally the upper band of two) and phenolise before ethanol precipitating the plasmid DNA.

Notes

Resuspending the DNA after ethanol precipitation may take a while, it is sometimes a good idea to leave pellet in the resuspension buffer for a while so as to maximise the yield.

Store large plasmids and cosmids in the fridge as they seem susceptible to damage when store in the freezer.

Carry out a normal plasmid prep to the phenolization step. Phenolize two or three times, chloroform once and ethanol precipitate. Spin down, wash and drain thoroughly. Dry the pellet fairly well, but complete dryness does not seem to be essential.

• Resuspend in 1.68ml of water and then add:

                                              0.32ml 5M NaCl
                                              2ml of 13% PEG 8000

• Mix thoroughly and leave on ice and water for more than 1h (overnight is possible).
• Spin down (10K; 20m; 4°C), wash, dry, resuspend in 250µl of water.
• Take samples.
• If the DNA seems free of RNA, make the solution 0.3M with NaCl, CHCl3 x 5 and etherize once with a 150µl 0.3 TNE followthrough.
• Ethanol precipitate, wash, dry and resuspend in 250µl of water.

Notes

If the DNA is very big, be prepared to dilute at stage 5, the solution can become very viscous if all of the PEG is not removed.

• Pick a single colony from a plate and inculate 250ml of LB broth containing the appropriate antibiotic. Grow the cells overnight in a shaker at 37°C.
• Centrifuge the cells at 5,000g, 4°C for 15m.
• Resuspend the cell pellet in 6ml of ice-cold freshly prepared lysis buffer:

                                                                         Lysis buffer

                                                                         25mM Tris.HCl, pH8.0
                                                                         10mM EDTA
                                                                         50mM glucose

• Thoroughly resuspend the cells by pipetting them up and down with a 10ml pipette. Incubate in ice water for 10m.
• Add 12ml of freshly prepared 0.2M NaOH/1% SDS. Carefully mix by inversion and incubate in ice water for 10m. Do not vortex.
• Add 7.5ml of 3M sodium acetate, pH4.6. Carefully mix by inversion or gentle vortexing and incubate in ice water for 20m.
• Centrifuge at 12,000 x g for 15m. Transfer the supernatant to another tube and discard the precipitate.
  Add DNAase-free RNAase A to a final concentration of 20µg/ml. Incubate at 37°C for 20m.
• Extract twice with 1 volume of TE-saturated phenol/chloroform. Vortex for 1m and centrifuge at 12,000g for 5m.
• Transfer the upper, aqueous phase to a fresh tube and add 1 volume of chloroform:isoamyl alcohol (24:1), vortex for 1, and centrifuge at 12,000g for 5m.
• Transfer the upper, aqueous phase to a fresh tube and add 2 volumes of ethanol. Leave at -20°C for 30m, then centrifuge at 12,000 x g for 20m.
• PEG purify.

You can expect about 500μg from this protocol.