GoldenGate Digestion-Ligation reaction (Level 1)

GoldenGate Digestion-Ligation reaction (Level 2)

Making LB Agar

Making LB broth

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

Making elctrocompetent Agrobacterium tumefaciens

Agrobacterium tumefaciens Electroporation Transformation

Blue- White Selection

Colony PCR

DNA mini-prep

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

Infiltration Analysis

Making GoldenGate compatible PSB1C3 'Flipper'

Antibiotic Selection