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Team:NRP-UEA-Norwich/Notebook Protocols
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Lab Protocols
GoldenGate Digestion-Ligation reaction (Level 1)
PROTOCOL HERE
GoldenGate Digestion-Ligation reaction (Level 2)
PROTOCOL HERE
Making LB Agar
PROTOCOL HERE
Making LB broth
PROTOCOL HERE
E. coli Calcium Chloride Heat Shock Transformation
Aim: to get DNA expression in E. coli.
- Remove chemically competent E. coli cells from the -80oC freezer and thaw on ice.
- Take 1-2 µL of DNA and transfer into a clean 1.5 mL tube.
- Add 50 µL of chemically competent E. coli to the DNA and incubate on ice for 30 mins.
- Preheat water bath to 42oC.
- Heat shock the DNA and E. coli tube for 30-60 sec (not more than 60 sec).
- Transfer back onto ice for 5 mins.
- Add 250-500 µL of LB broth to the tube and incubate at 370C with shaking for 2 hrs.
- Spread plate 100 µL onto plates containing the relevant antibiotics and incubate over night at 37oC.
E. coli Electroporation Transformation
PROTOCOL HERE
Making elctrocompetent Agrobacterium tumefaciens
PROTOCOL HERE
Agrobacterium tumefaciens Electroporation Transformation
PROTOCOL HERE
Blue- White Selection
PROTOCOL HERE
Colony PCR
PROTOCOL HERE
DNA mini-prep
PROTOCOL HERE
Preparation for sequencing
Aim: to sequence our DNA in order to check content of our constructs.
- Add 5µl of DNA sample at a concentration of 80-100ng/µl of Plasmid DNA (Diluting with sterile water if Plasmid DNA is at a concentration greater than 100ng/µl) to two separate 1.5 ml Eppendorf tubes.
- Add 5µl of primer 1 (Sense) to the first tube at a total concentration of 5µM (5pmol/µl) and 5µl of primer 2 (AntiSense) to the second tube (at a total concentration of 5µM (5pmol/µl)
- Send off both 10µl samples for sequencing.
Agrobacterium tumefaciens Infiltration
PROTOCOL HERE
Infiltration Analysis
PROTOCOL HERE
Making GoldenGate compatible PSB1C3 'Flipper'
PROTOCOL HERE
Antibiotic Selection
PROTOCOL HERE
