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From 2011.igem.org

Notebook: Week 4

Monday

- Made glycerol stock of Canada cells.

- Made plates 1mM IPTG + Amp

- Made 1 L LB, completed 1/40 dilution of ZeAFP (added Amp), shook for 3 hrs, checked OD until .78 OD reached

- Divided 1 L into 6 separate 100 mL solutions, half with IPTG, induced for 5 hours

- Centrifuged 4 100 mL solutions, saved supernatant/pellet for 2 (frozen at -20); for the other 2, we completed the following protocol:

1) Washed with 50 mL water

2) Resuspend in 100 mL ice-cold water

3) Divided 1ml aliquots into 8 eppendorf tubes (half induced half uninduced)

4) Added 100 and 10ul to IPTG and non IPTG plates (4 total) and grew at room temperature. Froze eppendorft tubes. Tomorrow we will unfreeze them for 6 hours on ice, then 1 hour at 4C, then replate to compare colony growth - essentially a survival assay after frozen : before frozen number of viable cells (this is obviously just a test assay)

- Plated 6 samples of the remaining 2 100 mL solutions, using 10 and 100 microliters on IPTG, LB, Amp plates and non-IPTG, LB, Amp plates and stored 1 of each concentration in 0 degrees, 4 degrees, and room temperature

Tuesday - Finished and presented project defense presentation to Sackler Institute and our advisers. - Made 3L worth of amp+iptg plates for large assay - Obtained TCA protein precipitation protocol: Stock solution 100% w/v Trichloroacetic acid (TCA). Recipie: dissolve 500g TCA (as shipped) into 350mL dH2O, store at RT. Protocol: add 1 volume of TCA stock to 4 volumes of protein sample. I.e. in a 1.5ml tube with maximum vol, add 250ul TCA to 1.0mL sample. Incubate 10min at 4C. Spin tube in microcentrifuge at 14K rpm, 5 min. Remove supernatant, leaving protein pellet intact. Pellet should be formed from whitish, fluffy ppt. Wash pellet with 200ul cold acetone. Spin tube in microcentrifuge at 14K rpm, 5 min. Repeat steps 4-6 for a total of 2 acetone washes. Dry pellet by placing tube in 95C heat block for 5-10min to drive off acetone. For SDS/PAGE, add 2X or 4X sample buffer (with or without BME) and boil sample for 10 min at 95C heat block before loading sample into Polyacrylamide gel. - Bacterial colonies for test of freezing assay are not growing yet - we will give more time b/c they were at room temperature previously. - Put induced and not-induced at 10 and 100ul on plates (the ones after frozen).

Wednesday

- Lawn of bacteria growing on RT plates. - OD measurements of frozen tubes to compare. Induced tubes have lower OD on average (statistically significant), likely due to protein expression. Need to compare % survival instead. - Interesting phenomenon whereby cells with anti-freeze protein in it forms elongated ice chunk while melting, but non-induced solution is fatter chunk. This is observed in eppendorf tubes containing the cells, but also in unfreezing supernatant. - Learned how to set a gel.

Stock solutions:

10X running buffer - bring to 1 Litres: - 144g glycine - 30g Tris base - 10g SDS Dissolve SDS to completion first. Heat at low after you put glycine adn Tris base in. Don't need pH

4x separating buffer bring to 100mls - 18.17g Tris base - 4.0 mls 10% SDS pH to 8.8 using concentrated HcL, then add dH2O to make up volume.

4x stacking gel buffer - 6.06g Tris-base - 10% SDS 2.0ml - 100mL H2O pH to 6.8 using conc H2O Ice morphology

Thursday

Plan:

Accomplished:

Friday

Plan:

Accomplished:

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