Team:Newcastle/Notebook/protocols

Contents 1 Streak plate 1.1 From glycerol stocks 1.2 From liquid culture: - 2 Liquid culture 3 Glycerol stocks 3.1 Making glycerol stocks 4 Chromosomal DNA Extraction 4.1 Nanodrop quantification of DNA extracts 5 PCR 6 QIAprep Spin Miniprep Kit 7 Making a culture of competent Basillus subtilis 168 7.1 Preparing MM competence media 7.2 Preparing starvation media

Streak plate

From glycerol stocks

1. Glycerol stock must be kept in an ice bucket.
2. Working close to lit Bunsen burner.
3. Flame a loop until red hot then dip into glycerol stock in cryo-vial.
4. Streak plate by: -
   1. Dragging loop across agar 5-10 times.
   2. Flame loop, then dip in agar (away from streak) to cool.
   3. Streak 4 times at a 90o angle from first streak.
   4. Streak again 4 times at 90o from second set of streaks.
   5. Streak again 4 times at 90o from third set of streaks to complete a square on the agar plate.
   6. Finally drag loop in a squiggle motion across the centre of the agar plate.
   7. The streak plate must then be left overnight at 37oC for colonies to grow.
   8. LB agar plates will usually be used unless alternative conditions are required.

From liquid culture: -

Protocol is similar to that of creating streak plates from glycerol stocks. However, an overnight culture of the strain to be streaked is grown and the loop is dipped into this culture rather than a cryo-vial of the glycerol stock.  

Liquid culture

Working close to lit Bunsen burner:

1. Liquid cultures can be grown in plastic universal tubes if only a small volume is needed (e.g. 5ml).
2. Transfer a colony from a plate of colonies using a flame-sterilised loop into liquid LB media in tube.
3. Cultures should be made from only one colony.
4. Leave the plastic universals on rotating plate (aerator) at 37°C overnight.

 

Glycerol stocks

Making glycerol stocks

NB: Only 50% glycerol solution is necessary for preserving cells by freezing at -80oC.

1. 1 volume of 100% glycerol should be mixed with 1 volume of Mili-Q filtered water (i.e. 1ml glycerol for 1ml water).
2. Vortex the solution briefly to mix.
3. Filter the solution using a sterile syringe filter, then pipette 500µl of the 50% glycerol solution into cryo-vials.
4. Pipette 500μl of the LB cell culture to be preserved into the cryo-vials.
5. Mix by inverting the tubes.
6. Glycerol stocks can then be stored in deep freeze at -80oC.

 

Chromosomal DNA Extraction

1. Add 1.75ml of bacterial cell culture to a 2ml tube.
2. Spin tubes at 20,000 x g (max speed of centrifuge may be 16,000 x g, if so spin at this speed) for 5 minutes in centrifuge and then discard the liquid supernatant.
3. Add 180μl of enzymatic lysis buffer (36μl lysozyme and 144μl buffer) to the tube and vortex for 10-20 seconds.
4. Incubate at 37oC for 20 minutes.
5. Add 25μl proteinase K.
6. Add 200μl Buffer AL.
7. Vortex briefly.
8. Incubate at 56oC for 5 minutes.
9. Add 200μl ethanol to the tube and vortex briefly.
10. Transfer entire contents (~600μl) to a spin column in 2ml collection tube using a pipette.
11. Centrifuge column at 10,000 x g for 1 minute.
12. Discard flow through and replace spin column in a 2ml collection tube.
13. Add 500μl Buffer AW1 to the column and centrifuge at 10,000 x g for 1 minute.
14. Discard flow through and replace spin column in a 2ml collection tube.
15. Add 500μl Buffer AW2 to the column and centrifuge at 16,000 x g for 3 minutes.
16. Carefully remove tubes from centrifuge without allowing the flow-through to contact the column (if this happens, spin tube in centrifuge again for 1 minute at 16,000 x g) and transfer column to a 1.5ml tube.
17. Add 200μl Buffer AE to the column and leave to stand at room temperature for 1 minute.
18. Centrifuge at 10,000 x g for 1 minute and then discard the column. Store the DNA in the collection tube at 4oC.

NB: Steps 17 and 18 can be carried out in repeat using smaller volumes of Buffer AE in order to try and increase DNA yield. Adding 30µl Buffer AE to the column and leaving to stand at room temperature for 5 minutes before centrifuging (and repeating 4-6 times) produces a high DNA yield.

 

Nanodrop quantification of DNA extracts

Nanodrop procedure:

1. Log into computer.
2. Open Nanodrop 1000.
3. Click on ‘Nucleic Acids’ button.
4. Default DNA setting will be DNA-50 – this is correct.
5. Wipe pedestal and top with tissue paper before starting.
6. Initialise by adding 3µl H2O to pedestal as instructed by the program.
7. Blank with 3µl elution Buffer (EB) (click ‘Blank’).
8. Add 3µl sample and click ‘Measure’.
9. Wipe pedestal with tissue paper between measurements.
10. Re-blank with water when finished to clean instrument, dry with tissue and log off.

PCR

1. Add the following reagents (quantities are for a 50µl reaction) to a PCR tube in the listed order:
   1. 27.5µl H2O
   2. 10.0µl 5x Buffer HF
   3. 1.0µl dNTP (200 µM)
   4. 5.0µl Reverse primer
   5. 5.0µl Forward primer
   6. 1.0µl Template DNA
   7. 0.5µl Phusion Polymerase (must be added last)
2. Mix these reagents by inverting the tube and then centrifuge for a couple of seconds for all of the reagents to collect at the bottom of the tube.
3. Set conditions of PCR on PCR machine:
   • 98°C 30 seconds initial denaturation
   • 98°C 10 seconds denaturation
   • x°C (10-20 seconds) anneal
   • 72°C 30 seconds/kb extension
   • 72°C 5-10 minutes final extension
   • 4°C hold
4. Store the PCR products at 4oC.

NB: The denaturation, anneal and extension steps are repeated cyclically for 30 cycles.

NB: Procedure for PCR differs between DNA polymerases. This procedure is for Phusion DNA polymerase.

QIAprep Spin Miniprep Kit

1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
2. Add 250 µl Buffer P2and mix by inverting the tube 4-6 times.
3. Add 350 µl Buffer N3 and mix immediately by inverting 4-6 times.
4. Centrifuge for 10 min at 13,000 rpm in a table top microcentrifuge.
5. Apply the supernatant to the QIAprep spin column by pipetting.
6. Centrifuge for 30-60s. Discard the flow-through.
7. Wash the QIAprep spin column by adding 0.5ml Buffer PB and centrifuging for 30-60s. Discard the flow-through.
8. Wash QIAprep spin column by adding 0.75ml Buffer PE and centrifuging for 30-60s.
9. Discard the flow-through, and centrifuge for an additional min to remove residual wash buffer.
10. To elute the DNA, put the QIAprep column in a clean 1.5ml microcentrifuge tube and add 50 µl Buffer EB to the centre of each QIAprep spin column. Let it stand for 1 min and centrifuge for 1 min.

Making a culture of competent Basillus subtilis 168

1. Inoculate 5ml of MM competence media with a single colony off the plate containing the strain in a 15ml falcon tube and incubate at 37 degrees and 180 rpm overnight

2. Next day, transfer 0.3ml of overnight culture into fresh 5ml MM competence media in a 50ml falcon tube and incubate for 3 hours at 37 degrees and 180 rpm

3. Add 5ml of pre-warmed starvation medium and incubate for a further 2 hours at 37 degrees and 180 rpm

4. Transfer 0.4ml of the culture to a 1.5ml microfuge tube and add 10µl DNA

5. Incubate for 1 hour at 37 degrees and 180 rpm – place tubes on sides to ensure maximum aeration

6. Plate out 200µl with appropriate antibiotic incubate over night at 37 degrees and 180 rpm

Preparing MM competence media

For 5ml you need:

• 5ml SMM media

• 62.5µl solution E (40% glucose)

• 50µl Tryptophan solution

• 30µl solution F

• 5µl casamino acids

• 2.5µl Fe-NH4-citrate

Preparing starvation media

For 5ml you need:

• 5ml SMM media

• 62.5µl solution E (40% glucose)

• 30µl solution F (MgSO4)