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

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(November 14, 2012)
(November 14, 2012)
Line 27: Line 27:
* 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'''
* 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'''
 +
* 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'''
-
{| class="wikitable" width=400px align="right"
+
{| class="wikitable" width=400px
|   || H2B || LOV  || Negative Control
|   || H2B || LOV  || Negative Control
|-
|-
-
| Insert DNA (X ng) || 1.28 μL || '''none'''
+
| Insert DNA (X ng) || 1.0 μL || 1.0 || 0
|-
|-
-
| Vector DNA (50 ng) || 5.0  || 5.0 || same
+
| Vector DNA (50 ng) || 5.0  || 5.0 || 5.0
|-
|-
-
| 2x Roche Rapid Ligation buffer || 5.0 μl || same
+
| 2x Roche Rapid Ligation buffer || 6.0 μl || 6.0 || 6.0
|-
|-
-
| New England Biolabs T4 ligase || 1.0 μl || same
+
| New England Biolabs T4 ligase || 1.0 || 1.0 || 1.0
|-
|-
| dH<sub>2</sub>O || ___ μL || ___ μL + ''Insert'' μL
| dH<sub>2</sub>O || ___ μL || ___ μL + ''Insert'' μL
|-
|-
-
| &nbsp; || 10.0 μL total &nbsp;&nbsp;|| same
+
| TOTAL || 12.0 || 12.0 || 12.0
|-  
|-  
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes.
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes.

Revision as of 22:42, 15 November 2012

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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 compared to vector: xμL H2B insert = 2 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 6.384 ng/μL H2B = 1.28 μL
  • 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
  H2B LOV Negative Control
Insert DNA (X ng) 1.0 μL 1.0 0
Vector DNA (50 ng) 5.0 5.0 5.0
2x Roche Rapid Ligation buffer 6.0 μl 6.0 6.0
New England Biolabs T4 ligase 1.0 1.0 1.0
dH2O ___ μL ___ μL + Insert μL
TOTAL 12.0 12.0 12.0
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 10 minutes.



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