Template:SBB-Protocols Assay1

Growth Assay Protocol

• Replate 16 constructs on new plates at the same time.

• As well, Plate DH10B, and pBca9495CA-Bca1144 (as controls, the DH10B is blank control and pBca9495CA-Bca1144 controls for plasmid effects.)

• Take 2 tubes of 280ul of cell and add 60ul KCM and 100ul water to each.

• Add 20ul of the KCM/water/cell solution into each construct.

• Transform (heat shock, etc.)

• Grow plates overnight.

• Pick 5 colonies from each sample.

• Grow to saturation in 96 well blocks with 400 uL LB media in each well.

• Then from each of the 5 unique liquid cultures, make an arabinose sample and a non-arabinose sample.

• Add 50 uL of LB media or LB Media+100ug/mL arabinose See note 1 below per well in 384 well plate.

• Add 1 uL of cell sample to each well.

• Place plate in Tecan and run OD measurements every 10 minutes.

Schedule

• For the first run-through, we'll do the 3 controls.

• Get one of the amazing GSI's to pick 5 colonies of each control Sunday night, suspending each colony in 400 uL LB media in 96 well plate.

Monday

• Run the assay, starting from the 384 well plate step.
• Plate all the constructs and the controls.

560 ul of cells (280*2) 2 tubes of cells
60ul of KCM
100ul H20

Tuesday

• Pick 5 colonies from each plate and grow on the 96 well block to saturation.

Wednesday

• Run the assay from the 384 well plate step.

JCA Notes

1. You should make cocktails of LB with whatever antibiotics or arabinose you need. Kanamycin and Chloramphenicol are stored at 25mg/mL. Ampicillin is stored at 100mg/mL. Arabinose is stored at 100mg/mL. At these concentrations, they are 1000x. You want to start with the same batch of LB for all samples in your plate. Calculate what total volume of each mixture of additives you'll need, and make up the mixtures in Eppendorf plates, mix well, then transfer the aliquots to the 384-well plates.

Follow up on Chris' notes:

So assuming everything is at 1000X concentrations, we need to dilute everytion to 1x in a 50ul LB media. This means that everything needs to be 1000x more dilute than it presently is. In order words we need just need to add a concentration that is 1000 times less than 50ul which is: 0.05ul of each in the 50 ul lb or 0.4ul of each in the 400ul lb

We can use the Tecan anytime after 1pm so we will need to get together as a group and decide that.

Lastly, for quantities of everything, we need (1/1000*400ul*19) or 7.6 ul of arabanose. We need (400ul*19) or 7.6ml (7600ul) of LB. All of our parts are CA so we need (1/1000*400ul*19) or 7.6 ul of both Ampicillin and Chloramphenicol.

So in short:
Chloramphenicol 6.8ul
Ampicillin 7.2ul
arabanose 7.6ul
LB 7.6ml

Plasmids DH10B (no antibiotics), pBca9145-Bca9494 (ampicilllin only), and pBca9495CA-Bca1144.
We don't need pBca9145-Bca9494 since it is only relevant for the strepavidin assay.
We ended up using 2 tubes of 280ul cells and 100ul of water and 60ul of KCM for each tube.