Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/12/06: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2012/12/06 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes)
 
(fix raw html notebook nav)
 
(7 intermediate revisions 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"|<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"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==Entry title==
==December 6, 2012==
* Insert content here...
----


'''Assemblies'''
* Final Gibson assembly products
# pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
# pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end
'''Dephosphorylation''' (11/23/12)
> Dephosphorylation (Roche)
# pSB1A3 - X/S = 2000 bp
{| class="wikitable" border="0" cellspacing="3" <!-- Dephos table -->
|-
| <u>Reagent</u> || <u>Volume</u>
|-
| DNA (clean digest) || 11.0 (~200 ng)
|-
| 10x phos. buffer || 1.5
|-
| phosphatase || 0.5
|-
| dH<sub>2</sub>O || 2.0
|-
| &nbsp; || 15 μL
|}
* Incubate at 37°C/ 10 min.
* Heat inactivate at 75°C/ 2 min.
* [final] = 200 ng/μL / 15 μL = ~13 ng/μL
'''Ligation'''
* Use 20 ng of vector for each ligation = '''1.5 μL pSB1A3'''
* Use 1x moles of H2B insert, relative to vector:  1xμL  H2B insert = 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 60 ng/μL H2B = at least '''0.07 μL H2B'''
* Use 1x moles of LOV insert, relative to vector:  1xμL  LOV insert = 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 62 ng/μL LOV = at least '''0.07 μL LOV'''
* Since the calulated amounts are so small and a volume of 5 μL is needed to add to the Gibson assembly mix, each value was multiplied by 2 and rounded for simplicity
{| class="wikitable" width=700px
| &nbsp; || H2B-LOV || LOV-H2B  || Negative Control
|-
| H2B-LOV H2B insert DNA (1x mol vector) || 1.0 μL || 0 || 0
|-
| H2B-LOV LOV insert DNA (1x mol vector) || 1.0 μL || 0 || 0
|-
| LOV-H2B H2B insert DNA (1x mol vector) || 0 μL || 1.0 || 0
|-
| LOV-H2B LOV insert DNA (1x mol vector) || 0 μL || 1.0 || 0
|-
| Vector DNA (39 ng) || 3.0  || 3.0 || 3.0
|-
| Gibson master mix || 15.0 || 15.0 || 15.0
|-
| Total volume || 20.0 || 20.0 || 18.0
|-
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at 50°C for 1 hour.
|}
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
[[Image:VN_Gibson_Assembly_Trial_1.png‎|thumb|frame|center|No colonies were seen the next day.]]
'''Made more pSB1A3 stock'''
{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
|-
|Trial || 260 || 280 || 260/280 || ng/μL
|-
|1 || 0.062|| 0.035 || 1.773|| 62.46
|-
|2 || 0.052|| 0.03 || 1.722|| 52.032
|-
|3 || 0.082|| 0.047|| 1.762|| 82.222
|-
|}


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Latest revision as of 22:19, 26 September 2017

Project name Main project page
Previous entry      Next entry

December 6, 2012

Assemblies

  • Final Gibson assembly products
  1. pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
  2. pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end

Dephosphorylation (11/23/12)

> Dephosphorylation (Roche)

  1. pSB1A3 - X/S = 2000 bp
Reagent Volume
DNA (clean digest) 11.0 (~200 ng)
10x phos. buffer 1.5
phosphatase 0.5
dH2O 2.0
  15 μL
  • Incubate at 37°C/ 10 min.
  • Heat inactivate at 75°C/ 2 min.
  • [final] = 200 ng/μL / 15 μL = ~13 ng/μL

Ligation

  • Use 20 ng of vector for each ligation = 1.5 μL pSB1A3
  • Use 1x moles of H2B insert, relative to vector: 1xμL H2B insert = 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 60 ng/μL H2B = at least 0.07 μL H2B
  • Use 1x moles of LOV insert, relative to vector: 1xμL LOV insert = 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 62 ng/μL LOV = at least 0.07 μL LOV
  • Since the calulated amounts are so small and a volume of 5 μL is needed to add to the Gibson assembly mix, each value was multiplied by 2 and rounded for simplicity
  H2B-LOV LOV-H2B Negative Control
H2B-LOV H2B insert DNA (1x mol vector) 1.0 μL 0 0
H2B-LOV LOV insert DNA (1x mol vector) 1.0 μL 0 0
LOV-H2B H2B insert DNA (1x mol vector) 0 μL 1.0 0
LOV-H2B LOV insert DNA (1x mol vector) 0 μL 1.0 0
Vector DNA (39 ng) 3.0 3.0 3.0
Gibson master mix 15.0 15.0 15.0
Total volume 20.0 20.0 18.0
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at 50°C for 1 hour.

Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101

No colonies were seen the next day.

Made more pSB1A3 stock

Trial 260 280 260/280 ng/μL
1 0.062 0.035 1.773 62.46
2 0.052 0.03 1.722 52.032
3 0.082 0.047 1.762 82.222


Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes/2012/12/06&oldid=1011667"