Template:SBB-Protocols Assay1

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Growth Assay Protocol

Replating 16 constructs and 2 controls on new plates concurrently.

The controls are: 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.

Transformation by Protocol

Competent cells are stored as 280uL aliquots in the -80 freezer as a communal stock.

  1. Thaw 2 tubes of 280 uL aliquot of cells on ice
  2. Add 100 uL of water to each tube
  3. Add 60 uL of KCM salts to each tube
  4. Add 1ul of the constructs to 20 uL of the cell cocktail. Pipette up and down gently to mix
  5. Let sit on ice for 10 min
  6. Heat shock for 2 min at 42
  7. Put back on ice for 1 min
  8. For ampicillin selection, you can plate immediately, otherwise:
  9. Add 100uL of LB, let shake in the 37 degree incubator for 40 min
  10. Plate on selective antibiotics, let incubate overnight

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.

Concentrations: 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.
  • We picked 1 colony each for the initial run and grew them up in 400ul of LB each

Wednesday

  • We made 2 tubes of LB. One 850ul tube with 0.85ul of arabanose and one with no added arabanose. We did not need to add antibiotics as it was premixed.
  • We added 50ul of LB (no arabanose) to 16 wells
  • We added 50ul of LB with arabanose to 16 different wells
  • In each of the wells, we added 1ul of the appropriate vector (11-26 or 16 in total)
  • We put our sample in the TECAN for 36 time points, 10 minutes between each.

Notes: we had a couple issues. First, the pipette wasn't totally accurate, a couple of us were short on LB from the 850ul tube even though we only needed 800ul. Also, we had some bubbles in the 384 well plate that, when they surface, splashed on the cover. Lastly, after inspection, looking at the side of the 384 well plate, we noticed that the levels of the fluid were not even at all. Some slots seemed to only have 1/2 of what they should be. Thank god this is the test run. I think for the actual run, we are going to take LESS rotations. Rotating between too many people for pippetting doesn't seem to help the accuracy of the measurement.

Monday 4-27-09

  • We are retransforming constructs 21-24 as well as controls DH10B and pBca9495CA-Bca1144 and replating.
  • Also we are restreaking the rest of the constructs 11-26 (minus 21-24 which failed the first time)

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.

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