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

Current revision

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The DNA was extracted from the gel slices using the Zymoclean Gel DNA Recovery Kit

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

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.

@@ Line 26: / Line 26: @@
 * 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
 | &nbsp; || 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