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Team:Washington/Protocols/test
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Revision as of 01:22, 12 September 2011
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
