User:Cassandra M Barrett/Notebook/Open Chromatin/2015/10/04
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Purpose: To build the following construct: https://benchling.com/s/x1UVKVF9/edit. This will be the first of many similar constructs, each with varying activation domains. ATF2 is the trial activation domain in this construct.
Begin by phosphorylating the parts. I will try two reactions, one with phosphorylated bb and one with unphosphorylated bb. Couldn't find buffer compatibility info between PNK and Ampligase but buffers appear very similar, won't clean up between phoshphorylation and LCR this time.
90fmol of each part was phosphorylated as follows:
Phosphorylation RXN 1 (set up two of these)
Incubate all three reactions at 37C for 30min. Inactivate at 65 for 20min
Set up three LCR reactions (30uL each)- one with phosphorylated MV10, one with unphosphorylated MV10, and one no bridges
Bridge1:LCRb_MV10tctGal4DB_rc Bridge2:LCRb_mCh_ATF2_rc Bridge3:LCRb_ATF2tcaMV10_rc
5 transformations: LCR1, LCR2, LCR3, H20 only control (-), MV10 plasmid control (+)