Team:NYMU-Taipei/Experiments/Protocols

Two-day cloning cycle

Labelling

Labelling tubes and plates in a consistent manner expedites the experimental process, and decreases the time taken to understand what others have done.

1.5ml microcentrifuge tubes

These 1.5ml microcentrifuge tubes are used to store extracted plasmids, cleaned up PCR or digested products, and frozen liquid culture.

Labelling:

• Black pen = Circular DNA, Blue pen = Linear DNA
• Folding part: Plasmid Backbone ("A" for pSB1A2 or "C" for pSB1C3 or "X" for other)
• On top (in this order):
  • Item name
  • Concentration (in ng/uL written in red)
  • "(PCR)" for PCR products; "(XP)","(SP)","(ES)","(EX)" for digestions
• On the side:
  • Date (YYYY-MM-DD),
  • Person who made it,
  • In red pen: the A260/280 and A260/230 ratios.
  • For Digestions, write the number of hours (or "overnight") it was digested.

200uL microcentrifuge tubes

200uL microcentrifuge tubes (or PCR tubes) are used as temporary storage of various reactions such as digestions, ligations, colony PCR, and temporary liquid cultures.

Labelling:

• Folding part: Write the day of the month.
• On top: Write something that can identify what it is (at least one english letter).
• Additionally:
  • Colony PCR tubes: Write everything in Black pen
  • Colony PCR LB tubes: Write everything in Blue pen
  • Digestion: Write the cutting site (e.g. SP,XP)
  • Ligation: Make sure you put it in those small bags for 4C overnight ligation after 1uL is taken out for transformation.

Agar plates

Agar plates are for transformations and obtaining single colonies.

• On top (agar side):
  • Full item names (not just numbers!), date (YYYY-MM-DD), time of incubation start.
  • Additionally for transformation plates: Name of person who did the plating.
• On side (still on agar side): type of antibiotic (A/Amp or C/CHL/CM).
• On bottom (non-agar side): Don't write anything here.

PCR

For any kind of PCR, make sure you list:

• the things that are PCR'd, and their expected PCR lengths.
• the PCR mix protocol: write the 50uL first, then to multiply to get the new mix total version.
• the PCR time protocol (the 94,[94,55,72]x30-35,72).
• how the gel was loaded. Which wells was loaded with which items.
• Which items were correct and are wrong after the gel run, in as much detail as possible (e.g. was the output the length of a self-ligated vector?)

Promoter Testing

These are the protocols we used for promoter testing

General Gene Reporter Assay protocol

This is our general setup for gene reporting assays.

Previous-night Preparation (~10m-60min)

Previous night:

• Culture overnight (~16hrs) at 37C and shaking at 180rpm 2-3ml of single colonies of things to be measured.

Preparation (can be done the previous night too):

• Determine the number of measurement points = number of items x number of replicates
• Prepare that amount of 15ml round-bottom tubes and 1.5uL microcentrifuge tubes.
• Add 2ml of LB + the relevant antibiotics in each round-bottom tube.

Pre-start (~30mins)

Preparation:

• Prewarm the LB+antibioics to the temperature to test at (e.g. 20mins prewarming in the incubator takes it up to 37C).
• Get some ice buckets and pre-cool down the 1xPBS

Culturing (2~3mins)

1. Add any relevant chemicals to the prewarmed round-bottom tubes containing 2ml LB.
2. Dilute the overnight culture to an OD600 of about 0.0325
3. Incubate at the required conditions (usually 37C and 180rpm).
4. Repeat for every replicate.

Measurement point retrieval

For each culture tube:

1. Take out the relevant culture and put it on ice
2. Extract 1.3ml from the culture tube and put it in an 1.5uL microcentrifuge tube
3. Centrifuge for 1min at 16.2g (13.2rpm on tabletop centrifuge)
4. WASH: Tip out the supernatant and add 650uL of 1xPBS, resuspend, then centrifuge again.
5. WASH again.
6. Carefully remove and discard all the supernatant, resuspend in 1.3ml of 1xPBS.
7. Store at 4C until measuring

Measuring

Requires black plates for measuring fluorescence and transparent plates for measuring OD600. For each measurement point, load 200uL each into 3 black wells and 3 transparent wells (i.e. 3 technical replicates each for fluorescence and OD600). Measure the fluorescence on the black plate with the settings depending on your machine. We use excitation/emission wavelength of 485/525 (100ms per measurement). Measure the OD600 on the transparent plate.

H₂0₂ Promoter Gene Reporter Assay

For measuring promoters under different H202 stress concentrations: (e.g.: 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM, 2mM, 2.5mM, 5mM).

• Add the H202 just before adding the bacteria.

For reporting assays using 2ml round-bottom tubes:

• First serially dilute the relevant concentrations from the stock (35%) everytime the experiments start.
• Add 13.4uL of the relevant concentration of H202 into the tube.