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

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(November 14, 2012)
(November 14, 2012)
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| pSB1A3|| 0.004 || 0.001 || 2.857 || 4.233
| pSB1A3|| 0.004 || 0.001 || 2.857 || 4.233
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|}
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----
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*'''[[User:Karmella Haynes|---Karmella]] 21:34, 15 November 2012 (EST)''': Next step - ligation & transformation<br>
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Refer to this page for a full description: http://openwetware.org/wiki/Haynes:Assembly101
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* Use 20 ng of vector for each ligation = 5 μL pSB1A3
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* 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'''
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{| class="wikitable" width=400px align="right"
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| &nbsp; || H2B || LOV  || Negative Control
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|-
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| Insert DNA (X ng) || 1.28 μL || '''none'''
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|-
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| Vector DNA (50 ng) || 5.0  || 5.0 || same
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|-
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| 2x Roche Rapid Ligation buffer || 5.0 μl || same
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|-
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| New England Biolabs T4 ligase || 1.0 μl || same
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|-
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| dH<sub>2</sub>O || ___ μL || ___ μL + ''Insert'' μL
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|-
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| &nbsp; || 10.0 μL total &nbsp;&nbsp;|| same
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|-
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| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes.
|}
|}

Revision as of 22:34, 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
  H2B LOV Negative Control
Insert DNA (X ng) 1.28 μL none
Vector DNA (50 ng) 5.0 5.0 same
2x Roche Rapid Ligation buffer 5.0 μl same
New England Biolabs T4 ligase 1.0 μl same
dH2O ___ μL ___ μL + Insert μL
  10.0 μL total    same
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 10 minutes.



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