Difference between revisions of "Team:UMaryland/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