Team:Georgia State/Collaborations

MA6b1. cells

centrifuged 2500 rpm for 5 or 10 min depending on the sample

LB supernatant was dumped

cells resuspended in 30 mL of 1500 uM phosphate buffer

centrifuged at 2500 rpm for 10 min

phosphate buffer supernatant dumped

cells resuspended in 32 mL of 1500 uM phosphate buffer

1 mL of resuspended cells put into 1.5 mL tubes labeled with the strain and time point

tubes centrifuged at 10k rpm for 5 mins at the specified time point

specified time points

t0= 30 mins

t2= 1 hr

t3= 2.5 hrs

t4= 24 hrs?

Leave overnight in a mixer at 37 C.

Next day, centrifuge 96 well plate.

Recipe for Malachite Green:

20 uL of the supernatant of each sample put into a new 96-well

60 uL of DI water added to each well with the sample to dilute

20 uL of Malachite green reagent added

Malachite Green -

500ul Malachite Green

125ul Ammonia

10ul Tween



Source

Source for picture of phosphate model: http://2017.igem.org/File:T--Georgia_State--Phosphate.png

MA6b1. cells

Day One Program

Take 5 mL of LB broth and place in 50 mL falcon tubes. Take a stab from glycerol stocks of two wild-types and two commercial strains(provided by Emory), place in tube. Let shake in the incubator overnight at 37 degrees.

centrifuged 2500 rpm for 5 or 10 min depending on the sample

LB supernatant was dumped

cells resuspended in 30 mL of 1500 uM phosphate buffer

centrifuged at 2500 rpm for 10 min

phosphate buffer supernatant dumped

cells resuspended in 32 mL of 1500 uM phosphate buffer

1 mL of resuspended cells put into 1.5 mL tubes labeled with the strain and time point

tubes centrifuged at 10k rpm for 5 mins at the specified time point

specified time points

t0= 30 mins

t2= 1 hr

t3= 2.5 hrs

t4= 5 hrs

t4= 7.5 hrs

t4= 24 hrs?

Leave overnight in a mixer at 37 C.

Next day, centrifuge 96 well plate.

Recipe for Malachite Green:

20 uL of the supernatant of each sample put into a new 96-well

60 uL of DI water added to each well with the sample to dilute

20 uL of Malachite green reagent added








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