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| - | {{:Team:UC-SantaCruz_Template}}
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| - | <p>
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| - | <strong>
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| - | 2013 UCSC IGEM TEAM Biofilm Membrane Notebook.
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| - | </strong>
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| - | </p>
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| - | <p>
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| - | The primary goal of the Biofilm Membrane team was to produce a high-density monolayer biofilm. A monolayer biofilm structure is critical to the function of
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| - | the biomachine as the overall goal of the project is to move salt across a membrane, producing potable water. Caulobacter Crescentus (C. crescentus), is a
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| - | polar body bacteria that forms high density monolayer biofilms and has been well characterized to support its use in biotechnological applications. The
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| - | biofilm forming capabilities of a strain of C. crescentus (CB15) have been well characerized by a group at Stanford University whose work we have cited.
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| - | CB15 was obtained from the Standford lab for the purpose of this project.
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| - | </p>
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| - | <p>
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| - | <strong>Week 1: To prepare materials that will be used for future experiment.</strong>
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| - | </p>
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| - | <p>
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| - | 8/15
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| - | </p>
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| - | <p>
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| - | <strong>Peptone Yeast Extract (PYE) Protocol</strong>
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| - | </p>
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| - | <p>
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| - | (for preparation of one Liter)
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| - | </p>
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| - | <p>
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| - | <strong>Aim</strong>
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| - | : To make 1 Liter of PYE growth medium for Caulobacter
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| - | </p>
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| - | <p>
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| - | <strong>Materials</strong>
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| - | :
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| - | </p>
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| - | <p>
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| - | Bacto Peptone 0.2% approx. 2g lot# 1194403
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| - | </p>
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| - | <p>
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| - | Yeast Extract 0.1% approx. 1g lot# 1307388 vat# 288620
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| - | </p>
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| - | <p>
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| - | MgSO4 1mM 0.247g EMD#MX0070-1 lot# uk09FZEMS
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| - | </p>
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| - | <p>
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| - | CaCl2 o.5mM 0.074g lot# A0141702020
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| - | </p>
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| - | <p>
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| - | 1 Liter flask
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| - | </p>
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| - | <p>
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| - | milliHQ water
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| - | </p>
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| - | <p>
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| - | autoclave
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| - | </p>
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| - | <p>
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| - | <strong>Procedure</strong>
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| - | :
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| - | </p>
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| - | <p>
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| - | 1) Combine 2g of bacto peptone, 1g of yeast extract, 0.24624g of MgSO4, and 0.07350g of CaCl2 in a sterile 1 Liter flask.
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| - | </p>
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| - | <p>
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| - | 2) Fill flask with millliHQ water to 1 Liter mark on flask.
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| - | </p>
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| - | <p>
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| - | 3) Homogenize mixture by shaking/stiring.
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| - | </p>
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| - | <p>
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| - | 4) Sterilize mixture using the autoclave on fluid cycle for 40 minutes.
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| - | </p>
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| - | <p>
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| - | 5) Let cool, cover, and store in refrigerator.
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| - | </p>
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| - |
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| - | <p>
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| - | Refer to
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| - | http://labs.mcdb.lsa.umich.edu/labs/maddock/protocols/media/antibiotics.html
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| - |
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| - | </a>
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| - | for Antibiotics Concentations.
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| - | </p>
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| - | <p align="right">
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| - | 4/9/93
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| - | </p>
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| - |
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| - | [[File:Antibiotic.png]]
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| - |
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| - | <p>
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| - | <strong>Notes:</strong>
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| - | </p>
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| - | <p>
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| - | Storage: All antibiotics should be stored at 4° C, Tet should be stored at -20° C.
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| - | </p>
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| - | <p>
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| - | Tet is also light sensitive. Store stock solutions & plates in the dark.
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| - | </p>
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| - | <p>
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| - | Prep: All antibiotics should be dissolved in sterile distilled water unless otherwise indicated.
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| - | </p>
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| - | <p>
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| - | Use oxytetracycline for Caulobacter strains.
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| - | </p>
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| - | <p>
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| - | <br\><br\>
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| - | 8/15
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| - | </p>
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| - | <p>
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| - | Innoculating Ecoli PxYFPC-2
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| - | </p>
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| - | <p>
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| - | <strong>Aim:</strong>
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| - | Picking Ecoli from a plate to get PxYPFC-2 plasmids
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| - | </p>
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| - | <p>
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| - | <strong>Materials:</strong>
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| - | </p>
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| - | <p>
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| - | -Ecoli PxYFPC-2 plate from Stanford
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| - | </p>
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| - | <p>
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| - | -LB growth medium (made by Aaron)
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| - | </p>
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| - | <p>
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| - | -Kanamycin ( stock conc. 50mg/ml)
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| - | </p>
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| - | <p>
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| - | -Inoculating Loop
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| - | </p>
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| - | <p>
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| - | -Flame
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| - | </p>
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| - | <p>
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| - | <strong></strong>
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| - | </p>
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| - | <p>
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| - | <strong>Procedure:</strong>
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| - | </p>
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| - | <p>
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| - | 1. Set up a flame
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| - | </p>
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| - | <p>
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| - | 2. Sterilize the loop by heating it over the flame
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| - | </p>
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| - | <p>
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| - | 3. Using the inoculating loop, pick a colony
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| - | </p>
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| - | <p>
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| - | 4. Dip loop into sterile LB broth (100ml)
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| - | </p>
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| - | <p>
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| - | 5. Store in shaking incubator at -37<sup>o</sup>C for 1 night
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| - | </p>
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| - | <p>
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| - | 8/19 Agar Plates
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| - | </p>
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| - | <p>
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| - | <strong>Aim:</strong>
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| - | To make PYE + antibiotic agar plates
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| - | </p>
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| - | <p>
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| - | <strong>Materials:</strong>
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| - | </p>
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| - | <p>
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| - | -Petri dishes
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| - | </p>
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| - | <p>
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| - | -PYE (500ml- this is enough to make 15-18 plates)
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| - | </p>
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| - | <p>
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| - | -Kanamycin/Gentamycin
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| - | </p>
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| - | <p>
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| - | -Agar (13-15 g per liter) [Lot # 2107450]
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| - | </p>
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| - | <p>
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| - | -1 Liter Erlenmeyer Flask
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| - | </p>
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| - | <p>
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| - | <strong></strong>
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| - | </p>
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| - | <p>
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| - | <strong>Procedure:</strong>
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| - | </p>
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| - | <p>
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| - | 1. Add 7.5g of agar and 500mL of PYE to a 1L flask.
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| - | </p>
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| - | <p>
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| - | 2. Homogenize mixture using a stirring bar.
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| - | </p>
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| - | <p>
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| - | 3. Autoclave for 40 mins on fluid cycle.
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| - | </p>
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| - | <p>
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| - | 4. Cool mixture to 50°C
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| - | </p>
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| - | <p>
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| - | 5. add antibiotic to mixture:
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| - | </p>
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| - | <p>
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| - | 6. For antibiotic concentrations refer to:
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| - | </p>
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| - | <p>
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| - | “A comprehensive set of plasmids for vanillate and xylose-inducible gene expression in Caulobacter Cresentus”
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| - | </p>
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| - | <p>
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| - | for kanamycin plates: add 250uL kanamycin
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| - | </p>
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| - | <p>
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| - | for gentamycin plates: add 250uL gentamycin
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| - | </p>
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| - | <p>
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| - | 7. Pour agar into sterilized petri dishes.
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| - | </p>
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| - | <p>
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| - | 8. Let stand for 15 mins to allow agar to solidify.
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| - | </p>
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| - | <p>
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| - | 9. Cover with parafilm and store in refrigerator.
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| - | </p>
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| - |
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| - |
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| - | <p>
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| - | <strong>Week 2: To create a standard growth curve for CB15</strong>
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| - |
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| - | </p>
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| - | <p>
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| - | 8/26
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| - | </p>
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| - | <p>
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| - | Ampicillin dilution
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| - | </p>
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| - | <p>
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| - | <strong>Aim:</strong>
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| - | to make stock ampicillin 50mg/ml
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| - | </p>
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| - | <p>
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| - | <strong>Materials:</strong>
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| - | </p>
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| - | <p>
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| - | -Ampicillin [Lot # 29371]
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| - | </p>
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| - | <p>
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| - | -BD syringe 0.2micron filtuer
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| - | </p>
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| - | <p>
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| - | -50ml centrifuge tube
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| - | </p>
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| - | <p>
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| - | -eppendorf tubes
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| - | </p>
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| - | <p>
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| - | <strong>Procedure:</strong>
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| - | </p>
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| - | <p>
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| - | 1. Weight out 0.5 g of Ampicillin
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| - | </p>
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| - | <p>
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| - | 2. Place in 50ml centrifuge tube along w/ 10ml of dH2O.
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| - | </p>
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| - | <p>
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| - | 3. Vortex the solution
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| - | </p>
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| - | <p>
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| - | 4. Using BD syringe filter out 10ml of diluted antibiotic into eppendorf tubes.
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| - | </p>
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| - | <p>
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| - | 5. Fill 20 eppendorf tubes w/ aliquot of 500ul.
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| - | </p>
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| - | <p>
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| - | 8/28
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| - | </p>
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| - | <p>
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| - | Growth Curve
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| - | </p>
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| - | <p>
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| - | <u>Aim</u>
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| - | : to create a standard caulobacter growth curve
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| - | </p>
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| - | <p>
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| - | <u>Materials:</u>
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| - | </p>
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| - | <p>
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| - | -PYE+AMP
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| - | </p>
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| - | <p>
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| - | -50ml centrifuge tubes x6
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| - | </p>
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| - | <p>
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| - | -CB15
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| - | </p>
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| - | <p>
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| - | <u>Procedure:</u>
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| - | </p>
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| - | <p>
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| - | <u>For 33 degrees C</u>
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| - | </p>
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| - | <p>
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| - | 1. Label 1 centrifuge tube “negative” and the other two c tubes. “33 ” and “33 b”
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| - | </p>
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| - | <p>
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| - | 2. Make 80ml of pye + amp.
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| - | </p>
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| - | <p>
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| - | Final conc of amp = 10ug/ml
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| - | </p>
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| - | <p>
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| - | Add 80ml of pye into a flask w/ 16ul of ampicillin( stock conc. 50mg/ml)
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| - | </p>
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| - | <p>
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| - | 3. Fill each tube w/ 4ml PYE
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| - | </p>
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| - | <p>
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| - | 4. For negative control tube dip a sterile pipette tip in the tube. Discard tip.
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| - | </p>
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| - | <p>
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| - | 5. For the other 2 tubes pick a colony from CB15 plate w/ a pipette tip. Pick two colonies one for each.
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| - | </p>
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| - | <p>
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| - | 6. Store in incubator at 33C at 300rpm
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| - | </p>
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| - | <p>
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| - | 7. Make a table and later transfer in unto a plot of O.D600 versus time graph.
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| - | </p>
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| - | <p>
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| - | 8. Record an OD reading every couple of hours.
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| - | </p>
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| - |
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| - | <p>
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| - | <br\>
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| - | <u>Results</u>
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| - | </p>
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| - |
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| - | [[File:Growth curves.png]]
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| - |
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| - |
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| - | <p>
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| - | 8/26
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| - | </p>
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| - | <p>
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| - | Inoculation of CB15
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| - | </p>
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| - | <p>
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| - | <strong>Aim:</strong>
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| - | making liquid culture of CB15
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| - | </p>
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| - | <p>
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| - | <strong>Materials: </strong>
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| - | </p>
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| - | <p>
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| - | -CB15(from Stanford)
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| - | </p>
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| - | <p>
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| - | -Antibiotic
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| - | </p>
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| - | <p>
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| - | -PYE
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| - | </p>
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| - | <p>
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| - | <strong>Procedure:</strong>
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| - | </p>
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| - | <p>
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| - | 1. Add 50ul of Ampicillin (50mg/ml) into 250ml of PYE
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| - | </p>
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| - | <p>
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| - | Refer to for final concentration of Ampicillin(10ug/ml):
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| - | </p>
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| - | <p>
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| - | <u>“Dynamics and Control of Biofilms of the Oligotrophic Bacterium Caulobacter Crecentus.”</u>
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| - | </p>
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| - | <p>
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| - | 2. Pipet 20ml of pye + amp into centrifuge tube. Do this twice. Label one “Negative” and the other “CB15”
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| - | </p>
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| - | <p>
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| - | 3. Pick a colony from CB15 plate and eject tip in tube
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| - | </p>
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| - | <p>
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| - | 4. For negative control, drop a blank pipet tip
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| - | </p>
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| - | <p>
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| - | 5. Incubate at 28°C and 180 rpm
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| - | </p>
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| - | <p>
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| - | <strong>Results:</strong>
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| - | </p>
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| - | <p>
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| - | After 1 night PYE medium for negative control turned cloudy. Experiment has been contaminated.
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| - | </p>
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| - | <p>
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| - | 8/29
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| - | </p>
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| - | <p>
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| - | Making Glycerol Stocks
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| - | </p>
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| - | <p>
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| - | <strong>Aim:</strong>
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| - | for long term storage of CB15
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| - | </p>
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| - | <p>
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| - | <strong>Materials:</strong>
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| - | </p>
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| - | <p>
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| - | -glycerol 60% (filtered)
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| - | </p>
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| - | <p>
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| - | -cell culture cb15
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| - | </p>
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| - | <p>
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| - | -eppendorf tubes.
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| - | </p>
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| - | <p>
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| - | <u></u>
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| - | </p>
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| - | <p>
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| - | <strong>Procedure:</strong>
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| - | </p>
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| - | <p>
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| - | 1. Prepare a preculuture: Pick a CB15 colony and inoculate it in 5ml of PYE. When culture reaches stationary phase the O.D600 should be around 1.3-1.6
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| - | </p>
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| - | <p>
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| - | 2. Caulobacters are store in 10% glycerol
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| - | </p>
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| - | <p>
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| - | Refer to: “Caulobacter S-Layer Secretion Kit Manual”
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| - | </p>
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| - | <p>
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| - | 3. Calculate 10% glycerol.
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| - | </p>
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| - | <p>
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| - | <u>(x amount of 10% glycerol</u>
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| - | )(60)= (900ul of cells + x)(10)
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| - | </p>
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| - | <p>
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| - | This comes out to be 180ul of 60% glycerol per 900ul of cb15
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| - | </p>
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| - | <p>
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| - | 4. In an eppendorf tube combine 180ul of 60% glycerol and 900ul of cb15 culture.
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| - | </p>
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| - | <p>
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| - | 5. Shake tube.
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| - | </p>
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| - | <p>
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| - | 6. Label and store tubes at -80C
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| - | </p>
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| - | <p>
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| - | 8/29-8/30
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| - | </p>
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| - | <p>
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| - | Preparing Electrocompetent CB15
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| - | </p>
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| - | <p>
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| - | <strong>Aim</strong>
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| - | : making electrocompetent CB15
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| - | </p>
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| - | <p>
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| - | <strong>Materials:</strong>
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| - | </p>
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| - | <p>
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| - | -PYE
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| - | </p>
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| - | <p>
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| - | -eppendorf tubes
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| - | </p>
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| - | <p>
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| - | -centrifuge tubes(50ml)
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| - | </p>
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| - | <p>
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| - | -1L falsk
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| - | </p>
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| - | <p>
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| - | -ice-cold, sterile, distilled water
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| - | </p>
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| - | <p>
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| - | -ice-cold, sterile 10% glycerol in distilled water.
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| - | </p>
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| - | <p>
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| - | <strong>Procedure</strong>
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| - | : will yield enough electro competent cells for 15 transformation.
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| - | </p>
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| - | <p>
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| - | 1. Diluting 60% stock conc. Glycerol into 10%
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| - | </p>
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| - | <p>
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| - | Add 80ml of distilled water and 16ml of 60% filtered glycerol from fridge into a flask
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| - | </p>
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| - | <p>
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| - | 2. Autoclave 1000ml of distilled water, 250ml PYE flask (x2), 96ml of 10% glycerol - fluid cycle 40mins
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| - | </p>
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| - | <p>
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| - | 3. Inoculate a CB15 colony into 5ml PYE medium. Grow at 30<sup>o</sup>C w/ shaking until the O. D is 0.5-1.0 ( should take about 1 night)
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| - | </p>
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| - | <p>
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| - | 4. Record the O.D of the culture above. <u>0.902</u>
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| - | </p>
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| - | <p>
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| - | 5. Dilution calculation: calculate the amount of culture you would add to 250ml of fresh PYE to achieve an O.D of 0.4-0.6 in 18hrs.
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| - | </p>
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| - | <p>
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| - | -doubling time of Caulobacter is about 3hrs. = 6 times doubling
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| - | </p>
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| - | <p>
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| - | 2<sup>6</sup> = 64 -> 0.5/64= 0.008(starting O.D)
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| - | </p>
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| - | <p>
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| - | (<u>x</u>ml)(0.902O.D)=(<u>250</u>ml)(.008O.D) x=2ml
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| - | </p>
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| - | <p>
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| - | Add 2ml of culture to 250ml PYE
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| - | </p>
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| - | <p>
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| - | 6. Incubate for 18hrs.
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| - | </p>
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| - | <p>
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| - | 7. Split 250ml of culture among five 50ml centrifuge tubes
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| - | </p>
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| - | <p>
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| - | 8. Centrifuge cells at 10000XG for 7mins at 4<sup>o</sup>C
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| - | </p>
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| - | <p>
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| - | 9. Remove supernatant and resuspend cells by in 50ml of ice-cold sterile, distilled water. Repeat for all tubes. DO NOT VORTEX
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| - | </p>
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| - | <p>
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| - | 10. Centrifuge w/ same conditions
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| - | </p>
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| - | <p>
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| - | 11. Remove supernatant and resuspend the cells in 25ml of ice-cold, sterile, distilled water.
| |
| - | </p>
| |
| - | <p>
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| - | 12. Centrifuge.
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| - | </p>
| |
| - | <p>
| |
| - | 13. Remove supernatant and resuspend the cells in 1/20 the original volume (50ml in each tube)
| |
| - | </p>
| |
| - | <p>
| |
| - | 1/20 * 50ml = 2.5ml of ice-cold, sterile 10% glycerol.
| |
| - | </p>
| |
| - | <p>
| |
| - | 14. Centrifuge
| |
| - | </p>
| |
| - | <p>
| |
| - | 15. Remove supernatant resuspend cells in 150ul of ice-cold, sterile 10% glycerol.
| |
| - | </p>
| |
| - | <p>
| |
| - | 16. Dispense cells into 50ul aliquot in eppendorf tubes. We ended up w/ 15 tubes total
| |
| - | </p>
| |
| - | <p>
| |
| - | 17. Store at 80<sup>o</sup>C.
| |
| - | </p>
| |
| - | <p>
| |
| - | 8/30
| |
| - | </p>
| |
| - | <p>
| |
| - | CB15 Transformation
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Aim</strong>
| |
| - | : Transforming CB15 w/ PxYFPC-2 and PvCFPC-4 plasmids
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Materials</strong>
| |
| - | :
| |
| - | </p>
| |
| - | <p>
| |
| - | -CB15(from Stanford)
| |
| - | </p>
| |
| - | <p>
| |
| - | -PxYFPC-2
| |
| - | </p>
| |
| - | <p>
| |
| - | -PvCFPC-4
| |
| - | </p>
| |
| - | <p>
| |
| - | -PYE+Kanamycin plates (x3)
| |
| - | </p>
| |
| - | <p>
| |
| - | -PYE+Gentamycin plates (x3)
| |
| - | </p>
| |
| - | <p>
| |
| - | -ice-cold sterile dH2O
| |
| - | </p>
| |
| - | <p>
| |
| - | -electroporation cuvettes (x3)
| |
| - | </p>
| |
| - | <p>
| |
| - | -sterile beads
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Procedure:</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | 1. If frozen, thaw electro competent cells on ice.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2. Place electroporation cuvettes on ice.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3. Dilution of Plasmids:
| |
| - | </p>
| |
| - | <p>
| |
| - | PxYFPC-2 conc. Is 293.2ng/ml in freezer.
| |
| - | </p>
| |
| - | <p>
| |
| - | -to dilute add 1ul of stock conc. into 100ul of sterile cold dh20. Making the final conc. 2.932ng/ul -> label this Px.
| |
| - | </p>
| |
| - | <p>
| |
| - | PvCFPC-4 conc. is 302.3 ng/ul in freezer
| |
| - | </p>
| |
| - | <p>
| |
| - | -to dilute add 1ul of stock conc. into 100ul of sterile cold dH20. Making the final conc. 3.023ng/ul -> label this Pv.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4. Pipet 100ul of electro competent CB15 cells to a electroporation cuvette.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5. Add 20ng of plasmid DNA so add 7ul of diluted plasmid DNA (PxYFPC-2)
| |
| - | </p>
| |
| - | <p>
| |
| - | 6. Mix around using pipette
| |
| - | </p>
| |
| - | <p>
| |
| - | 7. Electroporate at 2.5kV
| |
| - | </p>
| |
| - | <p>
| |
| - | 8. Record time constant.
| |
| - | </p>
| |
| - | <p>
| |
| - | 9. Take cuvette and immediately add 600ul of PYE. Mix using pipette.
| |
| - | </p>
| |
| - | <p>
| |
| - | 10. Transfer to microcentrifuge tube. Then to a shaking incubator at 33<sup>o</sup>C / 300rpm/ for 2hrs(we only left it in there for 1hrs)
| |
| - | </p>
| |
| - | <p>
| |
| - | 11. Repeat steps 5-11 for the second plasmid DNA (PvCFPC-4) and negative control. Instead of using plasmid DNA add 7ul of sterile dH20 for negative
| |
| - | control.
| |
| - | </p>
| |
| - | <p>
| |
| - | -Time constants a. pxyfpc-2 5.0
| |
| - | </p>
| |
| - | <p>
| |
| - | b. pvcfpc-4 5.2
| |
| - | </p>
| |
| - | <p>
| |
| - | b. negative 4.8
| |
| - | </p>
| |
| - | <p>
| |
| - | time constant b/w 3.6-4.3 msec are good (depends on the machine)
| |
| - | </p>
| |
| - | <p>
| |
| - | 12. Plating:
| |
| - | </p>
| |
| - | <p>
| |
| - | Plating scheme:
| |
| - | </p>
| |
| - | [[File:Plating scheme.png]]
| |
| - |
| |
| - | <p>
| |
| - | 13. For each tube of 600ul aliquot of transformed cells put 300ul in Kan plate and the other 300ul into Gent plate. Repeat this procedure 2 more times for
| |
| - | negative control and the other transformation.
| |
| - | </p>
| |
| - | <p>
| |
| - | 14. Incubate plates at 33<sup>o</sup>C for 3 days.
| |
| - | </p>
| |
| - | <p>
| |
| - | 9/2 (Monday)
| |
| - | </p>
| |
| - | <p>
| |
| - | After 3 days of incubation, results are as follows
| |
| - | </p>
| |
| - | [[File:CB15 transformation results.png]]
| |
| - | <p>
| |
| - | -There are contaminants in the negative control.
| |
| - | </p>
| |
| - | <p>
| |
| - | -Plates w/ transform cells turned out pretty well. The results are as expected. Since PxYFPC-2 is kanamycin resistant and PvCFPC-4 is gentamycin resistant,
| |
| - | transformation w/ PxYFPC-2 has growth only on PYE+KAN plate and PvCFPC-4 has growth on PYE+GENT plate.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Week 3: Production of Biofilms</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | 9/3
| |
| - | </p>
| |
| - | <p>
| |
| - | Production of Biofilms (Draft)
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Aim:</strong>
| |
| - | To grow high density biofilms.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Materials: </strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | -glass coverslips x6
| |
| - | </p>
| |
| - | <p>
| |
| - | -filter paper x6
| |
| - | </p>
| |
| - | <p>
| |
| - | -12 well culture plate
| |
| - | </p>
| |
| - | <p>
| |
| - | -pye+ amp
| |
| - | </p>
| |
| - | <p>
| |
| - | -preculture – prepared on 9/2.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Procedure</strong>
| |
| - | :
| |
| - | </p>
| |
| - | <p>
| |
| - | 1. Check O.D of preculture made on 9/2 by Rolando. 0.D = 1.250
| |
| - | </p>
| |
| - | <p>
| |
| - | Preculture: a colony was inoculated into a test tube w/ 5ml pye.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2. Autoclave glass coverslips (20min sterilization, 20 min dry)
| |
| - | </p>
| |
| - | <p>
| |
| - | 3. Flame forcep and prepare 100ml PYE + AMP(20ul)
| |
| - | </p>
| |
| - | <p>
| |
| - | 4. Cut filter paper into 2 pieces per sheet ( cut out 8 pieces)
| |
| - | </p>
| |
| - | <p>
| |
| - | 5. Place filter paper/glass in wells.
| |
| - | </p>
| |
| - | <p>
| |
| - | -modified design w/ 7 wells with filter paper and 5 wells with glass.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6. Fill each wells with 2ml PYE+AMP
| |
| - | </p>
| |
| - | <p>
| |
| - | 7. Inoculate 10 wells w/ 10ul of preculture culture and use 2 wells for negative control (one for glass and one for filter paper)
| |
| - | </p>
| |
| - | <p>
| |
| - | 8. Incubate at 33<sup>o</sup>C at 350rpm.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Results</strong>
| |
| - | : Images are obtained from bright field microscope under 40x after 20-24 hrs. of growth. There are no visible cells on filter paper(left) oppose to a semi
| |
| - | uniform layer of biofilm forming on the glass surface(right)
| |
| - | </p>
| |
| - | [[File:Biofilm 1.png]]
| |
| - | <p>
| |
| - | 9/6
| |
| - | </p>
| |
| - | <p>
| |
| - | Inducing xylose and vanillate
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Aim:</strong>
| |
| - | To inducing transform cells w/ 0.03% xylose or .5mM vanillate on agar plates.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Materials:</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | -pye+gent agar plate(x2)
| |
| - | </p>
| |
| - | <p>
| |
| - | -pye+kan agar plate(x2)
| |
| - | </p>
| |
| - | <p>
| |
| - | -1% xylose
| |
| - | </p>
| |
| - | <p>
| |
| - | -50mM vanillate
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Procedure:</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Plating scheme:
| |
| - | </p>
| |
| - | [[File:Plating scheme 1.png]]
| |
| - | <p>
| |
| - | 1. Calculate the amount of 1% xylose to add on agar plates
| |
| - | </p>
| |
| - | <p>
| |
| - | - Xylose = 0.03%
| |
| - | </p>
| |
| - | <p>
| |
| - | 15ml(0.03g/100ml) = x (.25g/25ml)
| |
| - | </p>
| |
| - | <p>
| |
| - | x = .45ml = 450ul
| |
| - | </p>
| |
| - | <p>
| |
| - | squirt 450ul of 1% xylose one pye + kan and one pye + gent plate. Wait 15mins until they dry
| |
| - | </p>
| |
| - | <p>
| |
| - | 2. Calculate the amount of 50mM vanillate to add on agar plates (pye +gent)
| |
| - | </p>
| |
| - | <p>
| |
| - | - Vanillate = 0.5mM
| |
| - | </p>
| |
| - | <p>
| |
| - | 50mM x = (15 + x )(0.5mM) x= 151.5ul
| |
| - | </p>
| |
| - | <p>
| |
| - | squirt 151.1ul of 50mM vanillate one pye+ gent and one + kan plate. Wait until they dry
| |
| - | </p>
| |
| - | <p>
| |
| - | 3. Pick a colony from CB15 + PxYFPC-2 transform plate and streak on a kan plate with xylose. Repeat for the other kan plate with vanillate.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4. Repeat step 3 for PvCFPC-4 on gent plates.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5. Incubate for 2 or 3 days at 33<sup>o</sup>C
| |
| - | </p>
| |
| - | <p>
| |
| - | 6. Look at plates under UV lamp
| |
| - | </p>
| |
| - | <p>
| |
| - | Results: There is no difference in fluorescent b/w induced and non-induced plates.
| |
| - | </p>
| |
| - | <p>
| |
| - | 9/5
| |
| - | </p>
| |
| - | <p>
| |
| - | Protocol for 50mM vanillate
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Aim:</strong>
| |
| - | to make 50mM vanillate from vanillic acid.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Materials:</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | -0.21g vanillic acid [lot b185020]
| |
| - | </p>
| |
| - | <p>
| |
| - | -naoh
| |
| - | </p>
| |
| - | <p>
| |
| - | -centrifuge tube
| |
| - | </p>
| |
| - | <p>
| |
| - | -.2 micron filter
| |
| - | </p>
| |
| - | <p>
| |
| - | -syringe
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Procedure:</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Calculation
| |
| - | </p>
| |
| - | <p>
| |
| - | X = (0.050)(0.025)(168.5) = 0.210 g vanillic acid
| |
| - | </p>
| |
| - | <p>
| |
| - | 1. Add 0.210g of vanillic acid acid into a centrifuge tube along w/ 25ml dh20.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2. Vortex solution. Until it homogenizes
| |
| - | </p>
| |
| - | <p>
| |
| - | 3. Titrate w/ NaOH until pH reaches 7.4
| |
| - | </p>
| |
| - | <p>
| |
| - | 9/9
| |
| - | </p>
| |
| - | <p>
| |
| - | Inducing xylose or vanillate into liquid cultures.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Aim:</strong>
| |
| - | Inducing xylose or vanillate into liquid cultures.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Materials: </strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | -PYE + Kan
| |
| - | </p>
| |
| - | <p>
| |
| - | -PYE+ Gent
| |
| - | </p>
| |
| - | <p>
| |
| - | -50mM vanillate
| |
| - | </p>
| |
| - | <p>
| |
| - | -50% xylose
| |
| - | </p>
| |
| - | <p>
| |
| - | -glass test tubes (x4)
| |
| - | </p>
| |
| - | <p>
| |
| - | -50ml centrifuge tubes (x4)
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Procedure:</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Calculations:
| |
| - | </p>
| |
| - | <p>
| |
| - | 2mM vanillate in 25ml PYE from 50mM stock concentration vanillate
| |
| - | </p>
| |
| - | <p>
| |
| - | X(50mM)= ( 10+x)(2) x= 412ul
| |
| - | </p>
| |
| - | <p>
| |
| - | 1% xylose in 10ml PYE from 50% stock concentration xylose.
| |
| - | </p>
| |
| - | <p>
| |
| - | X(50mM)= ( 10+x) x= 204ul
| |
| - | </p>
| |
| - | <p>
| |
| - | 1. Add 5ml of PYE+Kan to a test tube, then inoculate it w/ pxyfpc-2 by picking a colony from transformed plate. Make a negative
| |
| - | </p>
| |
| - | <p>
| |
| - | 2. Repeat step 1 for pvcfpc-4. Use pye + gent instead of pye + kan
| |
| - | </p>
| |
| - | <p>
| |
| - | 3. Incubate at 33<sup>o</sup>C 300rpm overnight.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4. Prepare 4 tubes, two w/ 10ml pye+kan and the rest w/ pye + gent.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5. For the two 10ml pye+kan inoculate each tube w/ 2.0ml of overnight preculture(pxyfpc-2). Do the same for pvcfpc-4 (2ml of preculture(pvcfpc-4) into
| |
| - | pye+gent)
| |
| - | </p>
| |
| - | <p>
| |
| - | 6. Wait a couple of hours until O.D reaches 0.4-0.6.
| |
| - | </p>
| |
| - | <p>
| |
| - | O.D 0.537 when induce
| |
| - | </p>
| |
| - | <p>
| |
| - | 7. Inoculate one pye+kan tube w/ 416ul of 50% xylose. The second one is negative control (Noninduced)
| |
| - | </p>
| |
| - | <p>
| |
| - | 8. Inoculate one pye +gent tube w/ 204ul of 50mM vanillate. Second one is noninduced
| |
| - | </p>
| |
| - | <p>
| |
| - | 9. Incubate at 33<sup>o</sup>C 300rpm overnight.
| |
| - | </p>
| |
| - | <p>
| |
| - | 10. Check if cells fluoresce under w/ U.V lamp.
| |
| - | </p>
| |
| - | <p>
| |
| - | Results: Induction w/ 2mM vanillate ( 2 tubes next to each other on the right) doesn’t seem to fluoresce under U.V light. On the contrary tube(far left) with 1% has a different color compare to its’ noninduced negative control
| |
| - |
| |
| - | </p>
| |
| - | [[File:Induction 1.png]]
| |
| - |
| |
| - | <p>
| |
| - | 9/10
| |
| - | </p>
| |
| - | <p>
| |
| - | 1X PBS (phosphate buffer saline) 1L
| |
| - | </p>
| |
| - | <p>
| |
| - | Aim: To make PBS for DAPI staining
| |
| - | </p>
| |
| - | <p>
| |
| - | Materials:
| |
| - | </p>
| |
| - | <p>
| |
| - | -8g of NaCl 137mM NaCl
| |
| - | </p>
| |
| - | <p>
| |
| - | -0.2g of KCl [lot 123796] 2.7mM KCl
| |
| - | </p>
| |
| - | <p>
| |
| - | -1.44g of Na2HPO4 [lot 116747] 10mM Na2HPO4
| |
| - | </p>
| |
| - | <p>
| |
| - | -0.24g KH2PO4 2mM KH2PO4
| |
| - | </p>
| |
| - | <p>
| |
| - | -HCl
| |
| - | </p>
| |
| - | <p>
| |
| - | Procedure:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1. Recipe above is for 1L. We’re only making 500ml so divide everything by 2.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2. Add 4g NaCl, 0.1g KCl, 0.72g Na2HPO4, and 0.12g KH2PO4 to a bottle
| |
| - | </p>
| |
| - | <p>
| |
| - | 3. Then add dH20 to the 500ml mark
| |
| - | </p>
| |
| - | <p>
| |
| - | 4. Titrate with HCl until pH reaches 7.4
| |
| - | </p>
| |
| - | <p>
| |
| - | 9/11
| |
| - | </p>
| |
| - | <p>
| |
| - | Stock solution of DAPI
| |
| - | </p>
| |
| - | <p>
| |
| - | Aim: to make stock solution of DAPI
| |
| - | </p>
| |
| - | <p>
| |
| - | Materials:
| |
| - | </p>
| |
| - | <p>
| |
| - | -10mg DAPI
| |
| - | </p>
| |
| - | <p>
| |
| - | -2ml dH2O
| |
| - | </p>
| |
| - | <p>
| |
| - | -20 eppendorf tubes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Procedure:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1. Add 2ml dH2O into 10mg DAPI vial.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2. Vortex vial.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3. Aliquot 10ul into eppendorf tubes.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4. Store in freezer. Away from light.
| |
| - | </p>
| |
| - | <p>
| |
| - | 9/11-9/12
| |
| - | </p>
| |
| - | <p>
| |
| - | Vanillate and xylose induction at different concentration.
| |
| - | </p>
| |
| - | <p>
| |
| - | Aim: to find a concentration at which transform cells fluoresce.
| |
| - | </p>
| |
| - | <p>
| |
| - | Materials:
| |
| - | </p>
| |
| - | <p>
| |
| - | -12 well culture plate (x3)
| |
| - | </p>
| |
| - | <p>
| |
| - | -CB15+PxYFPC-2 preculture (from previous experiment)
| |
| - | </p>
| |
| - | <p>
| |
| - | -CB15+PvCFPC-4 preculture (from previous experiment)
| |
| - | </p>
| |
| - | <p>
| |
| - | -CB15+PvCFPC-4 pick from a plate
| |
| - | </p>
| |
| - | <p>
| |
| - | -PYE+KAN
| |
| - | </p>
| |
| - | <p>
| |
| - | -PYE+GENT
| |
| - | </p>
| |
| - | <p>
| |
| - | -50% xylose
| |
| - | </p>
| |
| - | <p>
| |
| - | -50mM vanillate
| |
| - | </p>
| |
| - | <p>
| |
| - | Procedure:
| |
| - | </p>
| |
| - | <p>
| |
| - | 9/11
| |
| - | </p>
| |
| - | <p>
| |
| - | 1. Precultures of CB15+PxYFPC-2 and CB15+PvCFPC-4 were prepared from previous induction experiment. 5ul of preculture was added to new 5ml PYE.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2. Pick a colony from pvcfpc-4 transformed plate. Prepare a negative.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3. Incubate at 200rpm 33<sup>o</sup>C
| |
| - | </p>
| |
| - | <p>
| |
| - | 9/12
| |
| - | </p>
| |
| - | <p>
| |
| - | 4. Put 2ml of PYE+KAN in 6 wells on a culture plate. Repeat twice for PYE+Gent on new plates. (one gent would from preculture and the other one from a new
| |
| - | colony picked from plate)
| |
| - | </p>
| |
| - | <p>
| |
| - | 5. Inoculate all 2ml PYE+Kan wells w/ 250ul of pxyfpc-2 preculture.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6. For Pye+Gent inoculate one plate w/ 250ul of pvcfpc-4 preculture and the other plate w/ preculure from colony picked from plate
| |
| - | </p>
| |
| - | <p>
| |
| - | 7. Incubate at 33<sup>o</sup>C 200rpm until O.Ds of all cultures are round 0.4-0.6. Then induce according to the following scheme.
| |
| - |
| |
| - | </p>
| |
| - | [[File:Biofilms.png]]
| |
| - | <p>
| |
| - | 8. Incubate at 200rpm 33<sup>o</sup>C for atleast 5 hrs.
| |
| - | </p>
| |
| - | <p>
| |
| - | 9. Look under uv lamp for floursecent
| |
| - | </p>
| |
| - | <p>
| |
| - | Results.
| |
| - | </p>
| |
| - | <p>
| |
| - | For PxYFPC-2 there seems to be fluorescent at 0.5% xylose.
| |
| - | </p>
| |
| - | <p>
| |
| - | For PvCFPC-4 cells fluoresce at 5mM to 7mM
| |
| - | </p>
| |
| - | <p>
| |
| - | Protocol for DAPI staining
| |
| - | </p>
| |
| - | <p>
| |
| - | Refer to
| |
| - | http://www.igsb.org/uploads/pdf/ProtocolDAPIStaining.pdf
| |
| - | </p>
| |
| - | <p>
| |
| - | <u>Aim:</u>
| |
| - | To stain biofilms w/ DAPI
| |
| - | </p>
| |
| - | <p>
| |
| - | <u>Materials:</u>
| |
| - | </p>
| |
| - | <p>
| |
| - | -4ml of 4% PFA(paraformaldehyde) look at protocol
| |
| - | </p>
| |
| - | <p>
| |
| - | -1X PBS: look at protocol
| |
| - | </p>
| |
| - | <p>
| |
| - | -4ml of 0.1% Triton-X-100: look at protocol
| |
| - | </p>
| |
| - | <p>
| |
| - | -4ml of DAPI(stock conc. 1ug/ml): look at protocol
| |
| - | </p>
| |
| - | <p>
| |
| - | -fume hood
| |
| - | </p>
| |
| - | <p>
| |
| - | -Nutator
| |
| - | </p>
| |
| - | <p>
| |
| - | -biofilm coverslip
| |
| - | </p>
| |
| - | <p>
| |
| - | -12 well culture plate
| |
| - | </p>
| |
| - | <p>
| |
| - | -paper towel and aluminum foil for disposal for PFA
| |
| - | </p>
| |
| - | <p>
| |
| - | <u>Procedure:</u>
| |
| - | </p>
| |
| - | <p>
| |
| - | 1. Make 4% PFA, 1X PBS, 0.1% Triton-X-100, and DAPI stock conc. 1ug/ml (prepare these by looking at protocols). Do all work w/ PFA in fume hood.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2. Using a new 12 well culture plate, fill a well up w/ 4ml of DH20.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3. Take a coverslip to be analyze, wash slip by transferring it to the well w/ 4ml of dH2O for 2mins w/ gentle shaking (in order to get rid of swarmer and
| |
| - | unattached cells.)
| |
| - | </p>
| |
| - | <p>
| |
| - | 4. Do this next step in fume hood, since we are going to work w/ PFA. Aspirate dH2O and replace medium w/ 4ml of 4% PFA. Leave coverslip in solution for 8
| |
| - | minutes and place plate on a nutator. Do not leave it in there for more than 10mins.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5. Aspirate 4% PFA medium. PFA is volatile so dumped liquid waste onto paper towels that should be wrapped in aluminum foil before disposal.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6. Wash cells by inoculating 4ml of 1x PBS into well for 5mins. Place plate on a nutator. Repeat this process 2 more times (wash cells w/ 1x PBS for a
| |
| - | total of 3 times.)
| |
| - | </p>
| |
| - | <p>
| |
| - | 7. Remove 1X PBS and add 4ml of 0.1% Triton-X-100 (to permeabilize cells). Leave coverslip in there for 10mins. Also place plate on a nutator.
| |
| - | </p>
| |
| - | <p>
| |
| - | 8. After 10mins, repeat step 6. Wash cells 3 times w/ 1X PBS (5mins/wash.)
| |
| - | </p>
| |
| - | <p>
| |
| - | 9. Stain cells by inoculating well w/ 4ml of DAPI for 5mins. Place plate on a nutator.
| |
| - | </p>
| |
| - | <p>
| |
| - | 10. Wash cells w/ 1X PBS for 5 mins.
| |
| - | </p>
| |
| - | <p>
| |
| - | 11. Coverslip is now ready to be analyzed
| |
| - | </p>
| |
| - | <p>
| |
| - | 12. See microscopy protocol.
| |
| - | </p>
| |
| - | <p>
| |
| - | 9/16-9/20
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Production of Biofilm</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Aim</strong>
| |
| - | : To grow a high density uniform monolayer under varying parameters. We will culture the bacteria for 4 days taking data points at various time intervals.
| |
| - | The cultures will be grown on glass cover slips, micro-filter paper, and plastic.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Materials</strong>
| |
| - | :
| |
| - | </p>
| |
| - | <p>
| |
| - | C. crescentus CB15 (pre-culture)
| |
| - | </p>
| |
| - | <p>
| |
| - | PYE + ampicillin
| |
| - | </p>
| |
| - | <p>
| |
| - | Clorox
| |
| - | </p>
| |
| - | <p>
| |
| - | Ethanol
| |
| - | </p>
| |
| - | <p>
| |
| - | Milli HQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | 12 Well Tissue Culture Plate x 10
| |
| - | </p>
| |
| - | <p>
| |
| - | 40 Glass coverslips 15mm
| |
| - | </p>
| |
| - | <p>
| |
| - | 40 Filter Paper 0.22 micron
| |
| - | </p>
| |
| - | <p>
| |
| - | 40 Plastic coverslips
| |
| - | </p>
| |
| - | <p>
| |
| - | forceps
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Protocol</strong>
| |
| - | :
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Confirm if supply of pre-culture is available.
| |
| - | </p>
| |
| - | <p>
| |
| - | Preparing a pre culture: Using a pipette tip pick a colony from CB15 plate (from Stanford) dip it in 5ml PYE+amp. Incubate this at 200rpm and 33degrees C
| |
| - | overnight.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Prepare glass cover slips by cleaning with w/ ethanol. There’s 10% ethanol in lab if not we can make 10% ethanol by adding 100ml of ethanol to 900ml
| |
| - | dH20. Squirt some ethanol on coverslips and wipe them away with KimWipes.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Next sterilize glass pieces, PYE, dH2O, and forceps by autoclave at 120°C for 40 mins on fluid cycle
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Sterilize plastic pieces by using ethanol and rinsing off w/ sterile dH20
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>See figure for layout (these steps done for each different surface type)</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Place a cover slip in each well of tissue culture plate. We will have a total of 10 culture plates testing 3 different parameters (temps, days, and
| |
| - | replacement of nutrients) See figure for experiment layout.
| |
| - | </p>
| |
| - | [[File:Biofilms 2.png]]
| |
| - | <p>
| |
| - | 6) Add 2mL of PYE to each well.
| |
| - | </p>
| |
| - | <p>
| |
| - | 7) Identify negative control wells.
| |
| - | </p>
| |
| - | <p>
| |
| - | 8) Before inoculating check OD600 of preculture.
| |
| - | </p>
| |
| - | <p>
| |
| - | Procedure for spectrometer: Turn on the spectrometer, there’s a switch on the back of the machine. Hit option 4 Cell Count. Zero the machine out w/ a blank
| |
| - | (do this by filling a cuvette w/ PYE, place it in the machine, and hit auto zero.) There should be an arrow on the cuvette, turn the arrow until it faces
| |
| - | left and place it in the spectrometer. Read numbers off the top right. It should say 600nm and 0.00. Next pipette out 200ul of culture into another
| |
| - | cuvette, place it in the machine, and read off the top right corner or press start and it’ll pop out a number (this is the OD at 600nm).
| |
| - | </p>
| |
| - | <p>
| |
| - | 9) Inoculate each well that is to receive culture with 10uL of pre culture (CB15)
| |
| - | </p>
| |
| - | <p>
| |
| - | 10) Incubate at 33°C at 300rpm in incubator
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>To take a data point</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | 11 to take a data point at the 24hr interval, remove the coverslip to be analyzed. Use sterile forcep to handle coverslip. Wash coverslip to remove swarmer
| |
| - | and unattached stalked cells by preparing a petri dish filled w/ 15ml PBS (can also use a well and fill it w/ 2ml dh20). Place coverslip in PBS water for 2
| |
| - | min with gentle shaking.
| |
| - | </p>
| |
| - | <p>
| |
| - | (Rough graph for planning. Data points will include pictures of different staining methods. Look at different a protocol for staining)
| |
| - | </p>
| |
| - | <p>
| |
| - | 28°C
| |
| - | </p>
| |
| - | <table border="1" cellspacing="0" cellpadding="0">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | 24 hrs. nutrient replacement
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | <p>
| |
| - | 24hrs
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | <p>
| |
| - | 48hrs
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | <p>
| |
| - | 72hrs
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | <p>
| |
| - | 96hrs
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | Glass
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | Porous
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | Plastic
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | No Nutrient Replacement
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | Glass
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | Porous
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | Plastic
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | 33°C
| |
| - | </p>
| |
| - | <table border="1" cellspacing="0" cellpadding="0">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | 24 hrs. nutrient replacement
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | <p>
| |
| - | 24hrs
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | <p>
| |
| - | 48hrs
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | <p>
| |
| - | 72hrs
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | <p>
| |
| - | 96hrs
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | Glass
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | Porous
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | Plastic
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | No Nutrient Replacement
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | Glass
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | Porous
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td width="112" valign="top">
| |
| - | <p>
| |
| - | Plastic
| |
| - | </p>
| |
| - | </td>
| |
| - | <td width="47" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="55" valign="top">
| |
| - | </td>
| |
| - | <td width="83" valign="top">
| |
| - | </td>
| |
| - | <td width="57" valign="top">
| |
| - | </td>
| |
| - | <td width="66" valign="top">
| |
| - | </td>
| |
| - | <td width="60" valign="top">
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | PYE+AMP
| |
| - | </p>
| |
| - | <p>
| |
| - | 13) return culture plate to incubator.
| |
| - | </p>
| |
| - | <p>
| |
| - | 14) analyze removed cover slip under light microscopy. <strong>(see protocol)</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | 9/23
| |
| - | </p>
| |
| - | <p>
| |
| - | Protocol for 0.1% Triton-X-100
| |
| - | </p>
| |
| - | <p>
| |
| - | <u>Aim:</u>
| |
| - | To make 0.1% Triton-X-100 for DAPI staining
| |
| - | </p>
| |
| - | <p>
| |
| - | <u>Materials:</u>
| |
| - | </p>
| |
| - | <p>
| |
| - | -100% Triton-X-100
| |
| - | </p>
| |
| - | <p>
| |
| - | -250ml of sterile PBS
| |
| - | </p>
| |
| - | <p>
| |
| - | -sterile glass bottle for 250ml solution
| |
| - | </p>
| |
| - | <p>
| |
| - | <u>Procedure:</u>
| |
| - | </p>
| |
| - | <p>
| |
| - | 1. Add 250ul of 100% Triton X-100 and 249.75ml of dH20 to a glass bottle
| |
| - | </p>
| |
| - | <p>
| |
| - | 2. Storage: Store in tightly closed containter in a cool well-ventillated (area around 25C).
| |
| - | </p>
| |
| - | <p>
| |
| - | MSDS: <a href="https://fscimage.fishersci.com/msds/24515.htm">https://fscimage.fishersci.com/msds/24515.htm</a>
| |
| - | </p>
| |
| - | <p>
| |
| - | <a href="http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/1/t8532pis.Par.0001.File.tmp/t8532pis.pdf">
| |
| - | http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/1/t8532pis.Par.0001.File.tmp/t8532pis.pdf
| |
| - | </a>
| |
| - | (storage info)
| |
| - | </p>
| |
"
