- Pellet 1 mL bacterial overnight culture 5 times ( using a 1.5 mL centrifuge tube) by centrifugation at max speed (13000 rpm) for 60 secs room temperature (15 - 25)
- Resuspend pellet in 250 µL Buffer P1
- Add 250 µLBuffer P2 and mix thoroughly by inverting the tube 4-6 times until solution becomes clear
- Do not allow reaction to proceed for more than 5 mins
- If using Lyse Blue, reagent, solution will turn blue
- Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times
- Centrifuge for 10 mins at 13000 rpm
- Apply 800 µLsupernatant from step 5 to Q1A prep 2.0 spin column by pipetting
- Centrifuge for 60 secs and discard flow through
- Wash the Q1A prep column with 750 µL Buffer PE
- Centrifuge for 60 secs and discard flow through
- Centrifuge for 60 secs to remove residual wash buffer
- To elute DNA, add 50 µL DDH2O to the center of Q1A prep column
- Let stand for 1 min and centrifuge for 1 min
- Repeat steps 12 and 13
Ligation
Materials:
- 2 µL PSBIA3 digest
- 2 µL upstream digest (pBAD/ sRNBC)
- 2 µLdownstream digest (miraculin / const_GFP)
- 1 µL T4 DNA ligase
- 2 µL T4 DNA ligase 10x rxn buffer
Procedure:
- Combine reagents in a clean PCR tube
- Let stand 10 mins at room temperature/ no heat kill
