Team:Northwestern/Protocol

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[[Image:LOGOBANNER1.jpg|936px|center|link=http://wikipedia.org|Tech Institute]]
<!--- The Mission, Experiments --->
<!--- The Mission, Experiments --->
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{|- style="background:#F5EFFB; color:#8B008B" cellpadding="1" cellspacing="1" border="1" bordercolor="#F5EFFB" 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:#4B088A;">Home</span>]]
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!align="center"|[[Team:Northwestern|<font color="#2B3856">'''Home'''</font>]]
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!align="center"|[[Team:Northwestern/Team|<span style="color:#4B088A;">Team</span>]]
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!align="center"|[[Team:Northwestern/Brainstorm|<font color="#000000">'''Brainstorm'''</font>]]
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!align="center"|[[Team:Northwestern/Project|<span style="color:#4B088A;">Project</span>]]
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!align="center"|[[Team:Northwestern/Team|<font color="#000000">'''Team'''</font>]]
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!align="center"|[[Team:Northwestern/Parts|<span style="color:#4B088A;">Parts</span>]]
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!align="center"|[[Team:Northwestern/Acknowledgements|<font color="#000000">'''Acknowledgements'''</font>]]
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!align="center"|[[Team:Northwestern/Notebook|<span style="color:#4B088A;">Notebook</span>]]
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!align="center"|[[Team:Northwestern/Project|<font color="#000000">'''Project'''</font>]]
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!align="center"|[[Team:Northwestern/Safety|<span style="color:#4B088A;">Safety</span>]]
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!align="center"|[[Team:Northwestern/SideProject|<font color="#000000">'''Human Practices'''</font>]]
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!align="center"|[[Team:Northwestern/Calendar|<span style="color:#4B088A;">Calendar</span>]]
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!align="center"|[[Team:Northwestern/Parts|<font color="#000000">'''Parts'''</font>]]
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!align="center"|[[Team:Northwestern/Protocol|<span style="color:#4B088A;">Protocol</span>]]
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!align="center"|[[Team:Northwestern/Notebook|<font color="#000000">'''Notebook'''</font>]]
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!align="center"|[[Team:Northwestern/Calendar|<font color="#000000">'''Calendar'''</font>]]
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!align="center"|[[Team:Northwestern/Protocol|<font color="#000000">'''Protocol'''</font>]]
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!align="center"|[[Team:Northwestern/Safety|<font color="#000000">'''Safety'''</font>]]
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!align="center"|[[Team:Northwestern/Links|<font color="#000000">'''Links'''</font>]]
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!align="center"|[[Team:Northwestern/References|<font color="#000000">'''References'''</font>]]
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!align="center"|[[Team:Northwestern/Media|<font color="#000000">'''Media'''</font>]]
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!align="center"|[[Team:Northwestern/Contact|<font color="#000000">'''Contact'''</font>]]
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'''Protocol'''
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{| align="center" border="0" width="75%"
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|
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=='''Protocols'''==
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http://ginkgobioworks.com/support/
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== '''DNA''' ==
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Formatting: http://meta.wikimedia.org/wiki/Help:Wikitext_examples
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[[Team:Northwestern/Protocol/PDNA|Prepping DNA from the kit plates]]
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OpenWetWare Protocols: http://openwetware.org/wiki/Protocols
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[[Team:Northwestern/Protocol/Quikchange (from primers to colonies!|Quikchange: Primers to Colonies]]
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[[Team:Northwestern/Protocol/Kit to Stock Plasmid|Kit to Stock Plasmid]]
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== DNA ==
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[[Team:Northwestern/Protocol/Ethanol Precipitation|Ethanol Precipitation]]
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[[Team:Northwestern/Protocol/New Part Design(PCR)|New Part Design]]
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===[[Prepping DNA from the kit plates]]===
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=='''Bacterial Work'''==
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===[[Quikchange (from primers to colonies!)]]===
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[[Team:Northwestern/Protocol/Transformation|Transformation]]
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KIT TO STOCK
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[[Team:Northwestern/Protocol/O/N Culture|Overnight Cultures]]
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1. With a pipette tip, punch a hole through the foil cover into the corresponding well to the Biobrick™-standard part that you want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to cross contamination between the wells.
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[[Team:Northwestern/Protocol/Preparation of Competent Cells|Preparation of Competent Cells]]
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2. Add 10uL of diH2O (deionized water)
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3. Pipette 1 or 2uL of the resuspended DNA Transformation into your desired competent cells, plate bacteria with the appropriate antibiotic* and grow overnight.
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Transformation
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[[Team:Northwestern/Protocol/LB Media|LB Media]]
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1. Thaw cells on ice and pipette 25uL aliquots into tubes on ice
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2. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
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o Note: If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
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3. Let sit for 5 minutes on ice.
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o Note: For better efficiency do at least 30 minutes
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4. Incubate cells for 45 seconds at 42C.
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5. Incubate cells on ice for 2 min.
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6. Add 200 uL LB (2XYT and SOC are also suitable for greater efficiency)
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7. Incubate for 1 hour at 37C (for amp you can incubate for 5-10 minutes if you are in a hurry, for other ABs, can probably get away with ~40 minutes)
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8. Spread 100-300 μl onto a plate made with appropriate antibiotic.
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9. Grow overnight at 37C.
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10. Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.
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[[Team:Northwestern/Protocol/Preparing Plates|Preparing Plates]]
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4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 18 hours.
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[[Team:Northwestern/Protocol/Glycerol Stocks|Glycerol Stocks]]
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5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.
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See Miniprep Manual
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=='''Assembly'''==
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==Bacterial Work==
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[[Team:Northwestern/Protocol/Restriction Enzyme Digests|Restriction Enzyme Digests]]
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===[[Transformation]]===
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[[Team:Northwestern/Protocol/Plasmid Construction|Plasmid Construction]]
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===[[O/N Culture]]===
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[[Team:Northwestern/Protocol/3A Assembly|3A Assembly]]
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===[[Preparation of Competent Cells]]===
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[[Team:Northwestern/Protocol/Ligations|Ligations]]
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===[[LB Media]]===
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[[Team:Northwestern/Protocol/Mini Prep|Qiagen Mini Prep]]
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===[[Preparing Plates]]===
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=='''Microscopy'''==
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==DNA==
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[[Team:Northwestern/Protocol/Confocal Microscopy|Confocal Microscopy]]
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===[[Restriction Enzyme Digests]]===
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[[Team:Northwestern/Protocol/Fluorescence Microscopy|Fluorescence Microscopy]]
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===[[Ligations]]===
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=='''Reagents'''==
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===[[Mini Prep]]===
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[[Team:Northwestern/Protocol/Reagents|Reagents]]
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=='''Cell Staining'''==
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[[Team:Northwestern/Protocol/Rhodamine-Conjugated Chitin Probe|Rhodamine-Conjugated Chitin Probe]]
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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"