Team:Washington/Protocols

From 2011.igem.org

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[https://2011.igem.org/Team:Washington/Alkanes/Protocols/Example_2 Example 2]
[https://2011.igem.org/Team:Washington/Alkanes/Protocols/Example_2 Example 2]
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[http://soslab.ee.washington.edu/igem/2011/index.php/Sequencing  Sequencing Instructions]
[http://soslab.ee.washington.edu/igem/2011/index.php/Sequencing  Sequencing Instructions]

Revision as of 02:52, 11 September 2011

INSERT INFO HERE... LOOK AT OTHER PAGES FOR EXAMPLES OF FORMATING AND INSERTING PICTURES. CLICK THE EDIT BUTTON ON THE UPPER LEFT SIDE OF THE PAGE AFTER YOU HAVE SIGNED IN.

Example 1

Example 2


[http://soslab.ee.washington.edu/igem/2011/index.php/Sequencing Sequencing Instructions]

[http://soslab.ee.washington.edu/igem/2011/index.php/KunkelMutagensis Kunkel Mutagensis]

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

[http://soslab.ee.washington.edu/igem/2011/index.php/PlateExpression 96 Well Plate Protein Expression]

[http://soslab.ee.washington.edu/igem/2011/index.php/DoubleDigest Double Digest]

[http://soslab.ee.washington.edu/igem/2011/index.php/Projects:Biofuels/AlkaneBiosynthesis Alkane Biosynthesis Protocol]

[http://soslab.ee.washington.edu/igem/2011/index.php/Protocols/1L_NiNTA_Expression_Purification Standard 1L Expression Purification]

[http://soslab.ee.washington.edu/igem/2011/index.php/Protocols/GeneAssembly Gene Assembly With Oligos]


Protocol Page

restriction digest 10 uL DNA

5uL buffer ( 2 for most, check NEB)

.5 uL BSA

1uL enzyme 1

1uL enzyme 2

water to 50 uL(32.5 uL, add first)

oligo assembly by PCR

resuspend oligos with water, amount of water= concentration(in nm)*10 in uL

make oligo mix with 5uL of each primer

PCR reaction: 1uL phusion

.5uL oligo mix

1uL first oligo

1uL last oligo

5uL buffer

1uL dnTP

dH20 to 50uL

Ligation

7uL insert

1uL vector

1uL T4 ligase buffer

1uL T4 ligase

incubate at 37C 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

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