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Protocols

Recipes

LB Media (300mL)

Typtone 4g
Yeast Extract 2g
NaCl 4g

Make up to 400mL with RO water in a 500mL Schott bottle. Autoclave to sterilize

LB Agar (400mL)

Tryptone 4g
Yeast Extract 2g
NaCL 2g
Bacto agar 8g

Make up to 400mL in a 500mL Schott bottle with RO water. Autoclave for 15 minutes to sterilize. When the bottle is cool to the touch, add Antibiotic and pour out using aseptic technique.

SOC media (400mL)

Tryptone 8g
Yeast extract 2g
5M NaCl 0.8mL
1M KCl 1mL
1M MgCl2 4mL
1M MgSO4 4mL
2M D-Glucose 4mL

Make up to 400mL with RO water in measuring cylinder. Autoclave to sterilize.

50x TAE buffer (1L)

Tris Base (MW=121.1) 242.4g
EDTA disodium salt 18.61g

Dissolve mixture in 700mL of distilled water,then add:

Glacial Acetic acid 57.1mL

Make up to 1L. Dilute to 1 x solution before use.

1x TAE buffer (1L)

50x TAE buffer 20mL

Make up to 1L. Can be reused a few times before discard.

TB Buffer (500mL)

KCL (MW=74.5513) 9.3g
CaCl2.2H2O (MW=147.0146 1g
PiPES (MW=302.4) 1.5g

Dissolve in 300mL of water. Adjust to pH 6.7 with KOH.

Dissolve 5.44g of MnCl2.4H2O (MW=197.9052) in 100mL of Milli-Q water

Then add gradually to the solution of the remaining components.

Bring to 500mL and filter sterilize with 0.22µm filter. Store indefinitely at 4 degree.

1% Agarose Gel

DNA grade Agarose 1g
1x TAE buffer 100mL

Heat up in microwave (~1 minute) to dissolve. Cool to touch before adding 2µl of GelRed.

2 X M9 media for Xylose utilizing E.coli (500mL)

4x M9 salt powder 11.28g
Milli-Q Water 400mL
100 X Trace elements 10mL
1M MgSO4 1mL
1M CaCL 2 0.1mL
Leucine 130mg

Autoclave, wait to cool down, then add the following filter sterilised ingredients

0.1 mg/mL Thiamine 1mL

Top up to 500mL with sterile RO water.

Experiment Protocools

Agarose Gel Electrophoresis

  1. Measure 1g of DNA grade agarose powder in a CLEAN 250mL flask.
  2. Add 100mL 1XTAE buffer to agarose power and microwave for 1-2 minute until completely dissolved. Do not have a lid on when microwaving! Note - it will be very hot. Use heat-proof gloves to handle.
  3. Wait until agarose solution is cool to touch, then add 2ul of Gel Red DNA stain for every 100mL. Pour the agarose mixture into the gel cassette (with both ends tapped & with well comb in place) and leave to set (can take more than 30 minutes to set).
  4. Once set, remove the tap from both ends of the gel and place the gel into electrophoresis bath and fill with 1 x TAE buffer. Carefully remove the comb.
  5. Load the molecular marker. This typically goes into the first and sometimes the last well. Make sure the DNA ladder has been diluted according to manufacturer’s instructions and load between 1 to 2uL.
  6. Load the plasmid or DNA products (eg digested plasmid or PCR product) into each well by using approx 10-30ng of DNA eg 1ul of PCR products is usually sufficient. Mix the DNA with 6x loading dye.
  7. Run gel at 90V for 1h, using the dye as indicator of the progress. The bands should run from the negative (Black) to positive (Red).
  8. Take a picture of the gel image using the GelDoc system in the laboratory.

Producing competent cells

  1. Using a sterile plastic loop, pick 10-12 large (2-3mm in diameter) colonies from the plate. Inoculate to 150mL of SOB medium in a 1L flask, and grow overnight at 18-22oC, 200-250rpm. Alternatively, set up a starter culture (2ml) overnight and inoculate the large scale in the morning. Grow at 37 C.
  2. A600 should be 0.2-0.8 when harvest. Preferably, cells should be in mid log phase with A600 ~ 0.5
  3. Remove the flask from the incubator and place on ice for 10 minutes. FROM THIS STEP, KEEP THE CELLS ON ICE AS MUCH AS POSSIBLE!
  4. Transfer the culture to a 15mL centrifuge tube and spin at 2500 x g for 10 min at 4oC
  5. Pour off and discard the supernatant, and immediately place the tube on ice.
  6. Resuspend your cells in 1mL of ice-cold TB buffer, make sure there are no clumps of cells left, but also treat your cells gently and keep them cold.
  7. Add ice-cold TB buffer to bring volume up to 1/5th of the original culture volume (~30mL in this case). Mix the tube by gently inverting 3 times.
  8. Incubate the tube on ice for 10 minutes.
  9. Centrifuge at 2,500 x g for 7 minutes at 4o, pour off the supernatant.
  10. Gently resuspend the cells in ~1/20th of the original culture volume of ice-cold TB buffer. NOTE: 1/20th is based on and OD600 of 0.5, so adjust volume accordingly. E.g. if the culture OD600 was 0.1 then resuspend in 1/100th of original volume.
  11. Pre-chill 1.5ml Eppendorf tubes on ice. Add 930µl of your cell suspension, keeping the remainder on ice in the 15mL tube.
  12. Add 70µl of DMSO to the 930µl of cell suspension. Mix gently by swirling, and place on ice.
  13. Aliquot 100µl of the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the tubes with: Date – Strain. Snap freeze with liquid nitrogen or dry ice. Store cells at -80oC.

Transformation of E. coli Competent Cells with Heat Shock

  1. Obtain competent cells from -80oC.
  2. Defrost, then return on ice immediately. Always keep cells on ice up till Step 4. 50µl is sufficient for 1 transformation. If you have 100ul aliquot, split into 2 tubes.
  3. Add 1-10µl of plasmid DNA/ ligation mix to each tube. Incubate on ice for 5-15 min.
  4. Heat shock in 42oC water bath for 30 seconds, then back on ice for 2 min.
  5. Add 500µl of SOC media to each tube, and incubate in the 37oC shaker for 1h (30min for plasmid or 1h for ligation mix).
  6. For each tube of cells, spread 50µl onto one LB plate with appropriate antibiotic, and 200µl onto a second plate, using aseptic technique. Remember to label your plate properly (Your name, sample name, cell line, antibiotic, date)
  7. Leave plates (with lid on) on bench or 37oC incubator to dry out before sealing with parafilm. Place your plate upside-down in the 37oC incubator.

PCR Cleanup

From MO BIO's UltraClean 96 PCR Clean-up Kit Handbook

  1. Shake to mix the SpinBind before use. Add 5 volumes of SpinBind for each volume of your PCR reaction i.e. add 500 l to a 100 l PCR reaction.
    (If your PCR reaction and SpinBind volume is too large to fit in the PCR plate, you can use a 0.5 ml Collection Plate.)
  2. Mix well by pipetting. If an oil overlay was used, you will now have two layers. The top layer is oil.
  3. Place a 0.5 ml Collection Plate under a Spin Plate.
  4. Transfer PCR/SpinBind mixture to the wells of the Spin Plate, while avoiding the transfer of oil.
  5. Seal the wells with a piece of Centrifuge Tape.
  6. Centrifuge the Spin Plate/ Collection Plate at 4500 x g for 3 minutes.
    Before centrifuging, ensure that the stacked plates will clear the rotor and centrifuge.
  7. Remove the Spin Plate and discard the liquid flow-through from the 0.5 ml Collection Plate by inverting into an appropriate waste receptacle.
  8. Remove Centrifuge Tape and replace the Spin Plate in the same 0.5 ml Collection Plate.
  9. Add 300 l SpinClean® to the wells of the Spin Plate. Seal Spin Plate with new piece of Centrifuge Tape.
  10. Centrifuge at 4500 x g for 3 minutes.
  11. Remove the Spin Plate and discard the liquid flow-through from the Collection Plate by inverting into waste receptacle.
  12. Replace the Spin Plate in the same 0.5 ml Collection Plate.
  13. Centrifuge again at 4500 x g for 6 minutes.
  14. Carefully transfer the Spin Plate to a Microplate.
  15. Remove Centrifuge Tape.
  16. Allow to air dry for 10 minutes at room temperature.
  17. Add 100 l of Elution Buffer (10mM Tris) provided or sterile water directly onto the center of the white spin filter membrane of the Spin Plate. The choice of using Tris or water at this point will not affect yield. DNA is more stable for storage in Tris.
  18. Seal the Spin Plate with new Centrifuge Tape and centrifuge for 3 minutes at 4500 x g.
  19. Remove the Spin Plate from the Microplate. Purified DNA is now in the Microplate. Seal the Microplate with Elution Sealing Mat provided. The DNA will be free of all reaction components such as primers, enzyme, salt, and dNTP’s. Store DNA at -20oC. DNA is now ready to use.

Restiction enzyme digestion

The amount of restriction enzyme you use for a given digestion will depend on the amount of DNA you want to cut.
By definition: one unit of enzyme will cut 1 µg of DNA in a 50 µL reaction in 1 hour.
Reactions are often performed with 0.2-0.5 µL of enzyme because it is difficult to pipette less volume than this.
A typical restriction digestion reaction for 1µg of DNA in 50µL could look like this:

See: nebiolabs standard protocool for restiction enzyme digests
Restriction enzymes MUST be placed in an ice bucket immediately after removal from the -20 oC freezer because heat can cause the enzymes to denature and lose their function. If you need to cut less than 1µg for a large number of plasmids with the same enzyme(s), you can create a "Master Mix". A Master Mix consists of all of the reaction components except for the DNA. Aliquot your DNA into individual tubes and then add the appropriate amount of Master Mix to each tube. This will save you time and ensure consistency across the reactions.

Mix reagents + DNA as above in a clean 1.5mL eppendorf tube
Mix gently by pipetting/flicking. Spin to get all contents to bottom of tube if needed.
Incubate the tube at appropriate temperature (usually 37 °C) for 1 hour. Always follow the manufacturer’s instructions.
*TIP* Depending on the application and the amount of DNA in the reaction, incubation time can range from 1hr to overnight. For diagnostic digests, 1-2 hours is often sufficient. For digests with >1 µg of DNA used for cloning, it is recommended that you digest for at least 4 hours.
*TIP* If you will be using the digested DNA for another application (such as a digestion with another enzyme in a different buffer), but will not be gel purifying it, you may need to inactivate the enzyme(s) following the digestion reaction. This may involve incubating the reaction at 70 °C for 15 mins, or purifying the DNA via a purification kit
To visualize the results of your digest, conduct agarose gel electrophoresis.

Agarose Gel Preparation

  1. Measure 1g of DNA grade agarose powder in a CLEAN 250mL flask.
  2. Add 100mL 1XTAE buffer to agarose power and microwave for 1-2 minute until completely dissolved. Do not have a lid on when microwaving! Note - it will be very hot. Use heat-proof gloves to handle.
  3. Wait until agarose solution is cool to touch, then add 2ul of Gel Red DNA stain for every 100mL. Pour the agarose mixture into the gel cassette (with both ends tapped & with well comb in place) and leave to set (can take more than 30 minutes to set).

Glycerol Stock Preparations

  1. Put 500μL of glycerol in the acyrogenic tube.
  2. Put 500μL of sample liquid culture in the acryogenic tube.
  3. Place in ice.

Plasmid Isolation

  1. Centrifuge 0.5-4 mL of E.coli culture for 60 seconds at 6000 x gravity. Discard the supernatant (liquid around solid).
  2. Resuspend the resultant pellet into a mixture of Suspension Buffer and RNase A. (RNase A has been added if the yellow area on the Suspension Buffer container has been ticked.)
  3. Add 250ul Lysis Buffer.
  4. Mix gently and incubate for 5 mins at room temperature
  5. Add 350ul chilled Binding Buffer.
  6. Mix gently, incubate for 5 minutes on ice, then centrifuge for 10 minutes at maximum speed.
  7. Discard the pellet; use the supernatant for next steps. Leaving some supernatant behind is fine - avoiding contamination with the pellet is more important.
  8. Transfer collected supernatant to High Pure filter tube.
  9. Centrifuge at maximum speed for 30-60 seconds.
  10. Discard flowthrough.
  11. Centrifuge, without flowthrough, again at 13,000g for 1 minute.
  12. Discard flowthrough.
  13. Add 700ul Wash Buffer II.
  14. Centrifuge at 13,000g for 1 minute.
  15. Discard flowthrough.
  16. Centrifuge, without flowthrough, again at 13,000g for 1 minute.
  17. Discard flowthrough.
  18. Add 100ul Elution Buffer.
  19. Centrifuge at 13,000g for 1 minute.

Nanodrop Sample Concentration

  1. Open nanodrop software.
  2. Choose “DNA”.
  3. Lift arm over pedestal, wipe water off pedestal and arm with a dry kimwipe, apply 2µl of blank solution (whatever your nucleotides are resuspended in), and gently close arm.
  4. Hit “Blank” and the machine will blank the machine.
  5. When it displays and flat graph and a quantitation near 0, lift arm over pedestal, wipe blank off pedestal and arm with a dry kimwipe, apply 2µl of sample solution, and gently close arm.
  6. Hit “Measure” and the machine will measure your sample.
    The program will give you:
    • a quantitation in ng/µl
    • a graph of the absorbance
    • 260/280 ratio
    • 260/230 ratio

Optical Density with Biophotometer

  1. Pipette 100 ul sample from Eppendorf tube and pipette into measurement cartridge.
  2. Insert cartridge into biophotometer. Make sure that the length of the well is parallel to the arrow.
  3. Close lid.
  4. Measure sample.
  5. Record results.
  6. Repeat for each sample.

Biomass Sugar Extraction

Purpose To extract the sugars from samples of researched biomass sugar sources in the encyclopedia which have high potential for usage. Thus determine sugar concentration, and validate the suitability of various sources.
Biomass Bread
Peach
Banana
Coconut Pulp
Coconut Water
Sawdust
Barley
Wheat grain
Sorghum grain
Sugar cane bagasse
Bamboo
  1. Measure out 3g of each biomass source
  2. Place each sample in a separate Shott bottle
  3. Label each Shott bottle with the biomass
  4. Add aqueous chemical treatment up to the 40ml mark of the bottle
    • Distilled Water for Bread
    • 1% HCl for Peach, Banana, Coconut Pulp, Coconut Water, Strawberry
    • 1% NaOH for Sawdust, Barley, Straw, Bamboo, Bark, Sifton Bush, Wattle, Wheat, Grain, Barley, Grain, Sorghum, Grain, Agave
  5. Using a spatula, cut into small pieces, crush and mix
  6. Autoclave for 30 minutes at 131oC, then cool down solution
  7. Add 60 ml 50mM citrate buffer to the autoclaved solution to reduce pH to 5 (with the exception of bread - N/A)
  8. Add 0.1g enzymes and incubate for 72 hours at 50 degrees Celsius under shaking (with the exception of bread - N/A)
  9. Filter solution with filter paper and collect the flow through
  10. DNS assay
  11. Store samples in plastic tubes in freezer

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