Protocols

This page lists the protocols we used for our experiments.

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.

Labelling:

• 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?)

Two-day cloning cycle

We used an efficient two day cloning cycle split into a "Heavy" day and a "Light" day.

Approximate Light day:

• 10am (5min): Plate->4oC
• 10:05am(~1hr): 3-in-1, put liquid and plate in fridge.
• Lunch
• After lunch (~1hr): Run Gel
• 5pm: Incubate

Approximate Heavy day:

• 10am (2-3hrs): Plasmid extraction & Digestion
• Lunch (while digesting)
• After lunch (2-3hrs): Gel Extraction/Purification + Ligation + Transformation
• 5pm: Incubate plate

Light Day

The light day consists of Colony PCR and liquid culture of colonies transformed from a previous day.

The 3-in-1

The 3-in-1 protocol consists of a combination of three things:

• Colony PCR
• Liquid culture
• 2nd time plate

Time needed: [10min + 1min/colony]

Preparation
0. First, count the number of colonies you want to check.

1a. Make the Colony PCR mix (we use Thermo' DreamTaq) with the mix amount slightly modified:

We aliquoted 15uL of PCR mix for each colony.

1b. Aliquot 20uL of LB medium in a PCR tube for each colony. Put on the LB rack

1c. Take out new plates from the fridge and divide them into smaller sections (upto 24). This is the second-time plate. Make sure to write the date on it. Cross out in red pen any sections you know is wrong after the Colony PCR check

Steps
In this order:

1. Select a colony using a tip or toothpick.
2. Dip it in a PCR tube and swirl it around.
3. Use the same tip and dip it in the LB culture and swirl it around.
4. Use the same tip and draw on the 2nd time plate.

PCR run protocol:

Making Gel for gel electrophoresis

1. Make either 100ml or 200ml of gel at at time and select a relevant percentage (e.g. 1%, 1.5%, or 2%)
2. Measure out the relevant percentage in agarose (e.g. 2g of agarose for 100ml of 2% gel).
3. Fill it up with 1xTAE buffer.
4. Microwave on low, constantly taking it out every so often to swirl and mix it. Keep microwaving until clear. Make sure it is completely clear
5. Cool to a temperature that is still hot but still can be held in your hand. If the gel gets too cold and starts to harden, start microwaving again until clear.
6. Add 5uL of Safe-seeing dye for every 100ml of gel after cooled to the correct tempearature. Mix it well by swirling
7. Pour it onto the molds quickly. Put a cover on it to block out the light.
8. Wait at least 1/2hr until using it.
9. Store in 4^o and away from light.

Gel electrophoresis

1. Select a relevant % agarose gel based on your own experience (general guide: all <1000: 2%, all >2000: 1% guide)
2. Load 5uL from each tube of Colony PCR, mix it with 1uL of 6x DNA Dye and put it in a well.
3. Load 3uL of marker into a well.
4. Ru