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

(Difference between revisions)

Revision as of 10:04, 8 December 2012

Project name

Assemblies

Dephosphorylation (11/23/12)

> Dephosphorylation (Roche)

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.

No colonies were seen the next day.

Revision as of 10:00, 8 December 2012 (view source) Vi Nguyen (Talk \| contribs) (→December 6, 2012) ← Previous diff		Revision as of 10:04, 8 December 2012 (view source) Vi Nguyen (Talk \| contribs) (→December 6, 2012) Next diff →
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	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.]]