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Kunkel Mutagensis

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

96 Well Plate Protein Expression

Alkane Biosynthesis media and extraction

Standard 1L Expression Purification

Gene Assembly With Oligos

Protocol Page

restriction digest 10 uL DNA

5uL buffer ( 2 for most, check http://www.neb.com/nebecomm/DoubleDigestCalculator.asp)

.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