Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/12/06: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(2 intermediate revisions by the same user not shown) | |||
Line 38: | Line 38: | ||
* [final] = 200 ng/μL / 15 μL = ~13 ng/μL | * [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 | {| class="wikitable" width=700px | ||
| || H2B || LOV || Negative Control || H2B || LOV | | || 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 ( | | 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. | |||
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at | |||
|} | |} | ||
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101 | Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101 | ||
[[Image:VN_Gibson_Transformation_Trial_1.png|thumb|frame|center|No colonies were seen the next day.]] | |||
Revision as of 07:04, 8 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)
Ligation
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
|