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) |
|||
Line 6: | Line 6: | ||
| 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. ##### --> | ||
== | ==December 6, 2012== | ||
* Insert | ---- | ||
'''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 | |||
|- | |||
| || 15 μL | |||
|} | |||
* Incubate at 37°C/ 10 min. | |||
* Heat inactivate at 75°C/ 2 min. | |||
* [final] = 200 ng/μL / 15 μL = ~13 ng/μL | |||
{| class="wikitable" width=700px | |||
| || H2B || LOV || Negative Control || H2B || LOV || Negative Control | |||
|- | |||
| Insert DNA (2x mol vector) || 1.0 μL || 1.0 || --- || 1.0 || 1.0 || --- | |||
|- | |||
| Vector DNA (50 ng) || 1.5 || 1.5 || 1.5 || 1.5 || 1.5 || 1.5 | |||
|- | |||
| 2x Roche Rapid Ligation buffer || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 | |||
|- | |||
| T4 ligase || 1.0 NEB || 1.0 NEB || 1.0 NEB || 1.0 Roche || 1.0 Roche || 1.0 Roche | |||
|- | |||
| dH<sub>2</sub>O || 1.5 || 1.5 || 2.5 || 1.5 || 1.5 || 2.5 | |||
|- | |||
| TOTAL || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 | |||
|- | |||
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes. | |||
|} | |||
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101 | |||
Revision as of 17:40, 6 December 2012
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
December 6, 2012Assemblies
Dephosphorylation (11/23/12) > Dephosphorylation (Roche)
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
|