Difference between revisions of "Team:UMaryland/protocols"
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Protocols | 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 | ||
| + | |||
Revision as of 22:22, 17 September 2015
