Team:Washington/Protocols

From 2011.igem.org

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'''restriction digest'''
 
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10 uL DNA
 
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5uL buffer ( 2 for most, check  http://www.neb.com/nebecomm/DoubleDigestCalculator.asp)
 
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.5 uL BSA
 
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1uL enzyme 1
 
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1uL enzyme 2
 
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water to 50 uL(32.5 uL, add first)
 
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'''oligo assembly by PCR'''
 
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resuspend oligos with water, amount of water= concentration(in nm)*10 in uL
 
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make oligo mix with 5uL of each primer
 
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PCR reaction:
 
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1uL phusion
 
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.5uL oligo mix
 
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1uL first oligo
 
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1uL last oligo
 
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5uL buffer
 
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1uL dnTP
 
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dH20 to 50uL
 
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'''Ligation'''
 
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7uL insert
 
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1uL vector
 
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1uL T4 ligase buffer
 
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1uL T4 ligase
 
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incubate at <s>37C</s> no.  **Note that NEB website recommends 16C - room temp for ligations, we do ours at RT and it works well.  1 hour.
 
'''Colony PCR with Green tag'''
'''Colony PCR with Green tag'''

Revision as of 22:13, 13 September 2011


General Protocols

General PCR Protocol

General Digestion Protocol

General Ligation Protocol

General Transformation Protocol

Colony PCR Protocol

Competent Cell Prep Protocol

Kunkel Mutagensis

[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of how Kunkel Mutagensis works]


Gene Assembly With Oligos

Computational Protein Design

Sequencing

Make It: Diesel Production Protocols

Alkane Biosynthesis media and extraction


Break It: Gluten Destruction Protocols

Cell Lysate Assay

Small Scale (50mL) Protein Expression and Purification


Make It: Magnetosome Protocols

Gibson Reaction

Gibson Purification

Gibson Vectors (pGB) protocols

Check out or add wiki design tools here: Wiki Design



Colony PCR with Green tag

Master mix(7ul):

1ul 10uM forword primer

1ul 10uM reverse Primer

5ul 2x Green tag

Cell water(3ul): Pick one colony from the plate and mix with 10ul of ddH2O to make 10ul of cell water

Reaction = Master mix(7ul) + Cell water(3ul) = 10ul per tube

Use program "Colony" & change the extention time (1min per kb)

Heat Shock Transformation

2 ul ligation

20 ul cells

Ice 20 minutes

Heat shock at 42C for 1 minute

Ice 2 minutes

Prepare 200 ul of TB (no anti) and transformed cells in culture tube

Incubate at 37C for 1 hour

Plate cells

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