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

Light Day

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

1. The 3-in-1

   First, count the number of colonies that you want to check. Then, do the following 3 things sequentially:

   • Liquid culture
   • 2nd time plate
   • Colony PCR

   (Use the same tip to add the template to these three things)

2. Make The Gel For Electrophoresis
3. Run Gel Electrophoresis To Check The Colony PCR Product

Heavy Day

The heavy day consists of:

1. Previously grown plasmid extraction
2. Plasmid PCR
3. Gel extraction
4. Digestion
5. Ligation
6. Transformation

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

   Item	uL
   Primer(Forward and reverse)	1
   dNTP (10mM)	1
   10x DreamTaq buffer	5
   Taq Polymerase	0.2
   ddH20	42.8
   Total	50

2. Select a colony using a tip or toothpick.
3. Dip it in a PCR tube and swirl it around.

   PCR run protocol

   Temperature	Time
   94℃	60s
   94℃	15s
   55℃	20s	30-35 cycles
   72℃	1kb/min + 5-10s
   72℃	300s

1. Aliquot 4 mL of LB medium in a centrifugal tube for each colony.
2. Use a tip and dip it in the LB culture and swirls it around.

Take out new plates, for second-time purpose, from the fridge and divide them into smaller sections. Label the date properly. Cross out in red pen if any section is wrong after the Colony PCR is checked.

1. Make 200mL of gel at a time and select a relevant percentage (e.g. 1%, 1.5%, or 2%)
2. Measure and calculate the relevant percentage in agarose (e.g. 1g of agarose for 100mL of 1% gel).
3. Fill it up with 1xTAE buffer.
4. Microwave in few seconds, constantly taking it out to swirl and mix it each time. Keep radiating until solution turn transparent. Make sure it is completely transparent
5. Cool down the temperature of solution but make sure there is still a warmness in the flask. If the gel gets too cold and starts to harden, reheat the solution by microwave oven. Make sure it is transparent
6. Add 5uL of Safe-seeing dye for every 100mL of gel after cooled to the fine 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 15-20 min until using it.
9. Store in 4°C refridgerator and away from light.

1. Select a relevant percentage agarose gel based on your own experience
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. Run in the 13x13cm box at 60V or 70V and 400mA for the desired amount of time.
5. View in the gel viewer machine.

Insert and backbone:

Backbone check:
To check if the backbone is cut.

Digest for at least 1hr;over 2hr is better. EcoRI doesn't have star activity when cut this way (even overnight)

We run insert digests and backbone digests with backbone check on a gel. Then use GeneAid's gel extraction kit. The modifications in the protocol include:

Warm EB to 60-70℃ before elution.

Always use the Gel (sequencing) protocol for gel extraction.

We use Thermo's and NEB's T4 Ligase:

Incubate at room temperature for 2hr then transform 1uL then put the remaining amount in a small bag and put it in 4℃ overnight in case the transformation fails and retransformation is required.

We majorly use commercial E. coli DH5α competent cells and BL21 competent cells we made by ourselves.

• Add 1uL of plasmid or ligation mix to 20 uL of competent cells.
• Put mixture on ice for 30 minutes.
• Heat shock at 42℃ for 1 min.
• Put the mixture back on ice for another 20 minutes.
• Add 200 uL of LB broth to repair the cell wall; incubate at 37℃ for 1.5 hr.
• Plate it on a relevant antibiotic plate.
• Incubate plate at 37℃ overnight.