Team:Washington/Protocols/test

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Revision as of 01:09, 12 September 2011

46x30px

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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