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Revision as of 11:55, 1 October 2018
Protocols
E.coli Protocols
Two-day Efficient Cloning Cycle
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.
-
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)
- Make The Gel For Electrophoresis
- Run Gel Electrophoresis To Check The Colony PCR Product
Heavy Day
The heavy day consists of:
- Previously grown plasmid extraction
- Plasmid PCR
- Gel extraction
- Digestion
- Ligation
- Transformation
Colony PCR (Thermo DreamTaq®)
- 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 - Select a colony using a tip or toothpick.
- 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
Plasmid Extraction
ballalalla
Plasmid Extraction
ballalalla
Plasmid Extraction
ballalalla