Protocols
Protocols
Miniprep:
Materials:
250 µL Buffer P1
250 µLBuffer P2
350 µL Buffer N3
750 µL Buffer PE
100 µL DDH2O
Procedure:
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
If using Lyse blue, the solution will turn colorless
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
Was 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
Place Q1A prep column in a clean 1.5 mL microcentrifuge tube
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:
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
Let stand 10 minutes at room temperature / no heat kill
Transformation
50 µL cells
2 µL DNA
incubate on ice for 30 minutes
heats shock at 42 for 30 seconds
incubate on ice for 5 minutes
ADD 1 mL SOC media
incubate for 1 hour at 37
plate 200 µL
incubate at 37
3A assembly
5 µL Cutsmart
.5 µL BSA
.5 µL upstream
.5 µL downstream
ADD H20 to 20 µL
place in thermocycler
37 for 30 mins
80 for 20 mins
Gel extraction:
cut out gel portions with scalpel
weigh gel slice in colorless tube. Add 3 volumes of QG buffer to 1 volume of gel
Incubate at 50 for 10 mins until gels slice dissolves. Mix by vortexing tube every 2-3 mins during incubation to help the gel dissolve
After the gel dissolves completely, check color of the mixture is yellow (it may be orange or violet)
Add 10 µL of 3M sodium acetate (ph 5.0) to make solution more yellow (regardless of current solution color)
THe color of solution relates to ph indicator in
