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

Agrobacterium tumefaciens Infiltration

Infiltration Analysis

Making GoldenGate compatible PSB1C3 'Flipper'

Antibiotic Selection