Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/02/11: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(fix raw html notebook nav)
 
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"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|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]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
|-
|-
| colspan="2"|
| colspan="2"|

Latest revision as of 22:41, 26 September 2017

Project name Main project page
Previous entry      Next entry

February 9, 2013

Golden Gate Assembly

  • Mediated assembly
    • The volume of purified DNA (x) needed to dilute in a final volume of 20 μL = length in bp ÷ measured ng/μL * 20 fmols/μL * 650 fg/fmol ÷ 1,000,000 fg/ng * 20 μL final volume
    • Formula: x = length in bp ÷ measured ng/μL * 0.013 * 20
DNA length (bp) ng/μL vol. dH2O
H2B 410 77.995 1.4 18.6
LOV 461 80.443 1.5 18.5
pSB1A3 2000 77.4 6.7 13.3


Reagent Assembly Control
20 fmol H2B 1.0 0
20 fmol LOV 1.0 0
20 fmol pSB1A3 1.0 1.0
10x T4 ligase buffer (Promega) 1.0 1.0
T4 ligase (NEB) 0.25 0.25
BsmBI 0.5 0.5
dH2O 5.25 7.25
Total vol. 10.0 10.0
  • Bacterial transformation
    • Add total volume (10.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0 mL tube.
    • Incubate on ice for 2 min., heat shock at 42°C for exactly 45 sec., immediately place on ice.
    • Add 800 μL sterile SOC medium.
    • Grow with shaking at 37°C for 30 min.
    • Pellet the cells at top speed in a microcentrifuge for 3 min. at room temp.
    • Discard the supernatant. Resuspend the cells in 100 μL LB + antibiotic.
    • Plate cells on pre-warmed LB agar + antibiotic. Grow overnight at 37°C. Quick-transormation (e.g., DH5α-Turbo) is not recommended



Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes/2013/02/11&oldid=1012535"