SBB10AssayTeam1-Notes

Potential Timeline

Day 1: make master mix for 25 constructs, 2 arabinose, 2 non-arabinose for each construct. and transform by heat shock, grow overnight.
Day 2: pick colony and grow to saturation with LB media.
Day 3: make arabinose and non-arabinose samples out of saturated bacteria mixture, apply antibiotics (K and C), grow.
Day 4: run TECAN with cell aliquot.
Day 2-5: repeat Day 1-4 for the rest of the constructs.

Contact

• Jenna's e-mail: jenna.kelleher@gmail.com
• Maz's email: fuzzybulldozer@gmail.com
• Jim's email: jimhsu@berkeley.edu, jimhsu77479@gmail.com
• Apple's email: xiaoyan207@gmail.com, xliu@berkeley.edu. phone # is 415 627 7226.

Parts list

Media:140L_parts1_032910.xls

Toxicity

Toxicity Protocol

Controls: DH10B (blank), and pBca????-Bca1144 (plasmid effects.)
Parts: 2 arabinose, 2 non-arabinose

• 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 6 colonies from each sample.
• Grow to saturation in 96 well blocks with 400 uL LB media in each well. (16 parts total per well)
• Then from each of the 6 unique liquid cultures, make 3 arabinose samples and 3 non-arabinose samples
• Add 50 uL of LB media or LB Media+100ug/mL arabinose See note below per well in 384 well plate.

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.
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
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), and pBca????-Bca1144. 

• 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 # wells
• We added 50ul of LB with arabanose to # different wells
• In each of the wells, we added 1ul of the appropriate vector
• We put our sample in the TECAN for 36 time points, 10 minutes between each.

Transformation

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

Questions

• What are the parts to analyze? Basically everything on the parts list. Expect to use 3-4 plates
• What plasmid are we using? Antibiotic selection markers? A R6K - Kanamycin - Chloramphenicol plasmid.
• Number of replicates to do? 2 is sufficient.

Expression

• Plate washer: improves data quality and reproducibility
• rbs-prepro - periplasmic - deoxycholic acid
• rbs - cytoplasmic - "Bugbuster"

Questions

• Tags to use? (i.e. Myc, 6His, etc). Everything. The ones I know of: Myc, His, HA, FLAG
• Antibodies on plate?
• Available primary antibodies?
• How to make this quantitative? How much protein DO you have?

General protocol

Sample Prep

• Combine cell aliquots, KCM, and water
• Transform (heat shock, etc.)
• Grow plates overnight
• Pick colonies from each sample
• Lyse cells (using deoxycholic acid or bugbuster)

Reagant inventory

http://spreadsheets.google.com/ccc?key=0AvbXytCFRyFCdFdnbW15cENsQll0ZUJpYXFhbWRrX3c&hl=en

ELISA

• Source: http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=3141
• Add cell lysate into wells on plate (with antibody)
• Incubate
• Wash off unbound lysate and rinse with PBS
• Block with BSA
• Wash with PBS
• Add anti (something) antibodies
• Incubate
• Wash with PBS
• Add 2nd antibody conjugated with strepavidin/HRP, incubate
• Wash with PBS
• Add detection solution, incubate for a specific time
• Add stopping solution
• Read absorbance with ELISA plate reader