Team:Northwestern/Protocol

From 2010.igem.org

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[[Image:LOGOBANNER1.jpg|936px|center|link=http://wikipedia.org|Tech Institute]]
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<!--- The Mission, Experiments --->
<!--- The Mission, Experiments --->
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{|- style="background:#FFFFFF; style="font-size: 87%;" color:#000000" cellpadding="1" cellspacing="1" bordercolor="#000000" width="97%" align="center"
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{|- style="font-size: 87%; background: #EFCDF8; color: #FFFFFF" cellpadding="1" cellspacing="1" bordercolor="#000000" width="75%" align="center"
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!align="center"|[[Team:Northwestern|<span style="color:#000000;">'''Home'''</span>]]
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!align="center"|[[Team:Northwestern|<font color="#2B3856">'''Home'''</font>]]
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!align="center"|[[Team:Northwestern/Brainstorm|<span style="color:#000000;">'''Brainstorm'''</span>]]
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!align="center"|[[Team:Northwestern/Brainstorm|<font color="#000000">'''Brainstorm'''</font>]]
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!align="center"|[[Team:Northwestern/Team|<span style="color:#000000;">'''Team'''</span>]]
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!align="center"|[[Team:Northwestern/Team|<font color="#000000">'''Team'''</font>]]
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!align="center"|[[Team:Northwestern/Acknowledgements|<span style="color:#000000;">'''Acknowledgements'''</span>]]
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!align="center"|[[Team:Northwestern/Acknowledgements|<font color="#000000">'''Acknowledgements'''</font>]]
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!align="center"|[[Team:Northwestern/Project|<span style="color:#000000;">'''Project'''</span>]]
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!align="center"|[[Team:Northwestern/Project|<font color="#000000">'''Project'''</font>]]
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!align="center"|[[Team:Northwestern/SideProject|<span style="color:#000000;">'''Side Project'''</span>]]
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!align="center"|[[Team:Northwestern/SideProject|<font color="#000000">'''Human Practices'''</font>]]
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!align="center"|[[Team:Northwestern/Parts|<span style="color:#000000;">'''Parts'''</span>]]
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!align="center"|[[Team:Northwestern/Parts|<font color="#000000">'''Parts'''</font>]]
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!align="center"|[[Team:Northwestern/Notebook|<span style="color:#000000;">'''Notebook'''</span>]]
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!align="center"|[[Team:Northwestern/Notebook|<font color="#000000">'''Notebook'''</font>]]
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!align="center"|[[Team:Northwestern/Calendar|<span style="color:#000000;">'''Calendar'''</span>]]
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!align="center"|[[Team:Northwestern/Calendar|<font color="#000000">'''Calendar'''</font>]]
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!align="center"|[[Team:Northwestern/Protocol|<span style="color:#000000;">'''Protocol'''</span>]]
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!align="center"|[[Team:Northwestern/Protocol|<font color="#000000">'''Protocol'''</font>]]
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!align="center"|[[Team:Northwestern/Safety|<span style="color:#000000;">'''Safety'''</span>]]
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!align="center"|[[Team:Northwestern/Safety|<font color="#000000">'''Safety'''</font>]]
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!align="center"|[[Team:Northwestern/Links|<span style="color:#000000;">'''Links'''</span>]]
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!align="center"|[[Team:Northwestern/Links|<font color="#000000">'''Links'''</font>]]
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!align="center"|[[Team:Northwestern/References|<span style="color:#000000;">'''References'''</span>]]
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!align="center"|[[Team:Northwestern/References|<font color="#000000">'''References'''</font>]]
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!align="center"|[[Team:Northwestern/Media|<span style="color:#000000;">'''Media'''</span>]]
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!align="center"|[[Team:Northwestern/Media|<font color="#000000">'''Media'''</font>]]
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!align="center"|[[Team:Northwestern/Contact|<span style="color:#000000;">'''Contact'''</span>]]
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!align="center"|[[Team:Northwestern/Contact|<font color="#000000">'''Contact'''</font>]]
|}
|}
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{| align="center" border="0" width="75%"
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|
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=='''Protocols'''==
== '''DNA''' ==
== '''DNA''' ==
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[[Team:Northwestern/Protocol/PDNA|Prepping DNA from the kit plates]]
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===[[Prepping DNA from the kit plates]]===
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[[Team:Northwestern/Protocol/Quikchange (from primers to colonies!|Quikchange: Primers to Colonies]]
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===[[Quikchange (from primers to colonies!)]]===
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[[Team:Northwestern/Protocol/Kit to Stock Plasmid|Kit to Stock Plasmid]]
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===[[Kit to Stock Plasmid]]===
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[[Team:Northwestern/Protocol/Ethanol Precipitation|Ethanol Precipitation]]
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===[[Ethanol Precipitation]]===
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[[Team:Northwestern/Protocol/New Part Design(PCR)|New Part Design]]
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===[[New Part Design(PCR)]]===
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=='''Bacterial Work'''==
=='''Bacterial Work'''==
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===[[Transformation]]===
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[[Team:Northwestern/Protocol/Transformation|Transformation]]
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===[[O/N Culture]]===
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[[Team:Northwestern/Protocol/O/N Culture|Overnight Cultures]]
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===[[Preparation of Competent Cells]]===
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[[Team:Northwestern/Protocol/Preparation of Competent Cells|Preparation of Competent Cells]]
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===[[LB Media]]===
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[[Team:Northwestern/Protocol/LB Media|LB Media]]
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===[[Preparing Plates]]===
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[[Team:Northwestern/Protocol/Preparing Plates|Preparing Plates]]
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===[[Glycerol Stocks]]===
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[[Team:Northwestern/Protocol/Glycerol Stocks|Glycerol Stocks]]
=='''Assembly'''==
=='''Assembly'''==
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===[[Restriction Enzyme Digests]]===
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[[Team:Northwestern/Protocol/Restriction Enzyme Digests|Restriction Enzyme Digests]]
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===[[Plasmid Construction]]===
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===[[3A Assembly]]===
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[[Team:Northwestern/Protocol/Plasmid Construction|Plasmid Construction]]
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===[[Ligations]]===
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===[[Mini Prep]]===
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[[Team:Northwestern/Protocol/3A Assembly|3A Assembly]]
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[[Team:Northwestern/Protocol/Ligations|Ligations]]
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[[Team:Northwestern/Protocol/Mini Prep|Qiagen Mini Prep]]
=='''Microscopy'''==
=='''Microscopy'''==
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===[[Confocal Microscopy]]===
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[[Team:Northwestern/Protocol/Confocal Microscopy|Confocal Microscopy]]
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===[[Fluorescence Microscopy]]===
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[[Team:Northwestern/Protocol/Fluorescence Microscopy|Fluorescence Microscopy]]
=='''Reagents'''==
=='''Reagents'''==
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===[[Reagents]]===
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[[Team:Northwestern/Protocol/Reagents|Reagents]]
=='''Cell Staining'''==
=='''Cell Staining'''==
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===[[Rhodamine-Conjugated Chitin Probe]]===
 
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==Methanol Fixation==
 
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# Rinse slide with ethanol and flame
 
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# Rinse slide with PBS and dry with KimWipe
 
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# Apply cell sample to microscope slide and let air dry
 
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# Submerge in -20C absolute methanol for 5-10 min
 
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# Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash)
 
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** '''Do not let cells dry for the rest of the procedure'''
 
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==Staining==
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[[Team:Northwestern/Protocol/Rhodamine-Conjugated Chitin Probe|Rhodamine-Conjugated Chitin Probe]]
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# Make 1:500 dilution of chitin probe in TBS (50ul of probe is used per slide, [49.9ul of 1X TBS]:[0.1ul of chitin probe] --> 1 slide)
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# Apply 50ul to the slide containing the sample
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# Allow stain to incubate overnight at RT in a dark room
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** ''To prevent stain from evaporating overnight'': Use a large glass petri dish. Soak a paper towel with PBS and place it at the bottom of the petri dish. Use 2 toothpicks to support the slide above the paper towel. This prevents any wicking action from occurring and pulling the stain off the slide.
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# Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash)
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===[[LIVE/DEAD® BacLight - Bacterial Viability Kit]]===
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[[Team:Northwestern/Protocol/Methanol Fixation|Methanol Fixation]]
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[[Team:Northwestern/Protocol/LIVE/DEAD® BacLight - Bacterial Viability Kit|Live/Dead Baclight]]
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|}

Latest revision as of 01:23, 27 October 2010


Tech Institute
Home Brainstorm Team Acknowledgements Project Human Practices Parts Notebook Calendar Protocol Safety Links References Media Contact

Protocols

DNA

Prepping DNA from the kit plates

Quikchange: Primers to Colonies

Kit to Stock Plasmid

Ethanol Precipitation

New Part Design

Bacterial Work

Transformation

Overnight Cultures

Preparation of Competent Cells

LB Media

Preparing Plates

Glycerol Stocks

Assembly

Restriction Enzyme Digests

Plasmid Construction

3A Assembly

Ligations

Qiagen Mini Prep

Microscopy

Confocal Microscopy

Fluorescence Microscopy

Reagents

Reagents

Cell Staining

Rhodamine-Conjugated Chitin Probe

Methanol Fixation

Live/Dead Baclight

Retrieved from "http://2010.igem.org/Team:Northwestern/Protocol"