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 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

Colony PCR (Thermo DreamTaq®)

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