# Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/14

(Difference between revisions)
 Revision as of 22:46, 15 November 2012 (view source) (→November 14, 2012)← Previous diff Current revision (22:48, 15 November 2012) (view source) (→November 14, 2012) (2 intermediate revisions not shown.) Line 26: Line 26: * Use 20 ng of vector for each ligation = '''5 μL pSB1A3''' * Use 20 ng of vector for each ligation = '''5 μL pSB1A3''' - * Use 2x moles of H2B insert compared to vector:  xμL  H2B insert = 2 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 6.384 ng/μL H2B = '''1.28 μL H2B''' + * Use 2x moles of H2B insert, relative to vector:  xμL  H2B insert = 2 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 6.384 ng/μL H2B = '''1.28 μL H2B''' - * Use 2x moles of LOV insert compared to vector:  xμL  LOV insert = 2 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 11.492 ng/μL LOV = '''0.8 μL LOV''' + * Use 2x moles of LOV insert, relative to vector:  xμL  LOV insert = 2 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 11.492 ng/μL LOV = '''0.8 μL LOV''' {| class="wikitable" width=400px {| class="wikitable" width=400px |   || H2B || LOV  || Negative Control |   || H2B || LOV  || Negative Control |- |- - | Insert DNA (X ng) || 1.0 μL || 1.0 || 0 + | Insert DNA (2x mol vector) || 1.0 μL || 1.0 || 0 |- |- | Vector DNA (50 ng) || 5.0  || 5.0 || 5.0 | Vector DNA (50 ng) || 5.0  || 5.0 || 5.0 |- |- - | 2x Roche Rapid Ligation buffer || 7.0 μl || 7.0 || 7.0 + | 2x Roche Rapid Ligation buffer || 7.0 || 7.0 || 7.0 |- |- | New England Biolabs T4 ligase || 1.0  || 1.0 || 1.0 | New England Biolabs T4 ligase || 1.0  || 1.0 || 1.0

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## November 14, 2012

• The DNA was extracted from the gel slices using the Zymoclean Gel DNA Recovery Kit
 Sample 260 280 260/280 ng/μL H2B 0.006 0.004 1.463 6.384 LOV 0.011 0.004 2.769 11.492 pSB1A3 0.004 0.001 2.857 4.233

• ---Karmella 21:34, 15 November 2012 (EST): Next step - ligation & transformation

Refer to this page for a full description: http://openwetware.org/wiki/Haynes:Assembly101

• Use 20 ng of vector for each ligation = 5 μL pSB1A3
• Use 2x moles of H2B insert, relative to vector: xμL H2B insert = 2 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 6.384 ng/μL H2B = 1.28 μL H2B
• Use 2x moles of LOV insert, relative to vector: xμL LOV insert = 2 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 11.492 ng/μL LOV = 0.8 μL LOV
 H2B LOV Negative Control Insert DNA (2x mol vector) 1.0 μL 1.0 0 Vector DNA (50 ng) 5.0 5.0 5.0 2x Roche Rapid Ligation buffer 7.0 7.0 7.0 New England Biolabs T4 ligase 1.0 1.0 1.0 dH2O 0 0 1.0 TOTAL 14.0 14.0 14.0 Mix the reaction(s) thoroughly by flicking the tube.Incubate at room temperature for 10 minutes.

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