IGEM:MIT/2006/Notebook/2006-6-23

To Do

1. Ligation reaction
2. Run gel of everybody PCR
3. Dilute (then induce at 4 hrs and 8 hrs) the overnight LB and EZ liquid cell cultures
   • time test for pleasant smells at intervals after IPTG induction
4. Repeat transformation of biobrick ligations into BL21 cells

Repeat ligation reaction

1. Remove Backbone/BSMT tube and Backbone/Control part tube from storage in -20c fridge
2. Considerations:
   • want 10 μL total volume
   • want 50 nanograms of both of the desired dnas in each ligation tube: ( i.e. backbone and BSMT -or- backbone and barry's control part)
   • for the spec readings remember that the amount of each desired dna will be HALF of the reading (b/c have 2 dnas in each tube), so make calculations appropriately
3. Ligation BSMT Test:
   • 1 μL T4 DNA ligase buffer (be gentle, also check that it smells like wet dog)
   • .5 μL T4 ligase enzyme
   • 7 μL of BSMT = 50 nanograms of backbone and BSMT pcr product (basically want 100 nanograms of 15.7ng/μL, where 15.7 is from dna spec reading)
   • 1.5 μL water (volume neccesary for 10 μL total volume)
4. Ligation Control Part Test:
   • 1 μL T4 DNA ligase buffer (be gentle, also check that it smells like wet dog)
   • .5 μL T4 ligase enzyme
   • 8 μL control = 50 nanograms of backbone and control part dna (spec = 13 ng/μL)
   • .5 μL water (volume neccesary for 10 μL total volume)
5. Thermocycle for 1.5 hrs at 16c with a 10 minute heat shock at 65c

Repeat transformation

1. See protocol here
2. Considerations:
   • use flat bottom tubes
   • heat shock 50 seconds
   • vortex control sample before transforming
3. Plate both 20 μL and 200 μL