User:Cassandra M Barrett/Notebook/Open Chromatin/2015/10/04: Difference between revisions
(→LCR) |
(fix raw html notebook nav) |
||
(One intermediate revision by one other user not shown) | |||
Line 2: | Line 2: | ||
|- | |- | ||
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
Line 93: | Line 93: | ||
*Add 750uL of SOC media (RT) to each tube | *Add 750uL of SOC media (RT) to each tube | ||
*Shake 37C for 1.5hrs | *Shake 37C for 1.5hrs | ||
*Plate on LB+Amp (Spin down, remove 200uL of supernatant, resuspend and plate) | *Plate on LB+Amp (Spin down, remove 200uL of supernatant, resuspend and plate 100uL) | ||
*Incubate overnight at 37C | *Incubate overnight at 37C | ||
Latest revision as of 01:13, 27 September 2017
Project name | Main project page Previous entry Next entry |
LCRPurpose: 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. Methods: 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 LCR Set up three LCR reactions (30uL each)- one with phosphorylated MV10, one with unphosphorylated MV10, and one no bridges LCR1:
LCR2:
LCR3:
Bridge1:LCRb_MV10tctGal4DB_rc Bridge2:LCRb_mCh_ATF2_rc Bridge3:LCRb_ATF2tcaMV10_rc
Transformation 5 transformations: LCR1, LCR2, LCR3, H20 only control (-), MV10 plasmid control (+)
|