Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/20

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(Autocreate 2012/11/20 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes)
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==Entry title==
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==November 20, 2012==
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* Insert content here...
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* Made liquid cultures of pSB1A3 and PT7CFE1
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* In a 30 μL reaction, cut a [http://partsregistry.org/Part:pSB1A2 pSB1A2 plasmid] (from Rene) XbaI and SpeI, then ran on 1% gel and used Zymoclean Gel DNA Recovery Kit
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{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
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|-
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| Reagent || pSB1A2
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|-
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| DNA || 15.0
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|-
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| XbaI || 1.0
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|-
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| SpeI || 1.0
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|-
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| 10x buffer || 3.0
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|-
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| dH<sub>2</sub>O || 10.0
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|-
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| Total vol. || 30.0
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|}
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<center>
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[[Image:VN_Gel_11-20-12.png|200px|Results of from the digests are shown.]]
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[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]
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</center>
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<center>
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Results of the enzyme digest is shown, with both pSB1A3 bands on the right. The gel was loaded with 30 μL samples.  The bands that were cut are outlined in green.
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</center>
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* Results from gel recovery
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{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
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|-
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| Sample|| 260|| 280|| 260/280 || ng/μL
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|-
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| pSB1A3|| 0.017|| 0.008|| 2.132|| 17.238
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|}
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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 = '''1 μL pSB1A3'''
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* 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'''
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* 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'''
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{| class="wikitable" width=400px
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| &nbsp; || H2B || LOV  || Negative Control
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|-
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| Insert DNA (2x mol vector) || 1.0 μL || 1.0 || 0
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|-
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| Vector DNA (50 ng) || 1.0  || 1.0 || 1.0
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|-
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| 2x Roche Rapid Ligation buffer || 7.0 || 7.0 || 7.0
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|-
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| New England Biolabs T4 ligase || 1.0  || 1.0 || 1.0
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|-
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| dH<sub>2</sub>O || 4.0 || 4.0 || 5.0
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|-
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| TOTAL || 14.0 || 14.0 || 14.0
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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.
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|}
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Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
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Revision as of 15:54, 21 November 2012

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November 20, 2012

  • Made liquid cultures of pSB1A3 and PT7CFE1
  • In a 30 μL reaction, cut a pSB1A2 plasmid (from Rene) XbaI and SpeI, then ran on 1% gel and used Zymoclean Gel DNA Recovery Kit
Reagent pSB1A2
DNA 15.0
XbaI 1.0
SpeI 1.0
10x buffer 3.0
dH2O 10.0
Total vol. 30.0

Results of from the digests are shown. Image:KAH_Fermentas_GeneRuler_1kbplus.jpg

Results of the enzyme digest is shown, with both pSB1A3 bands on the right. The gel was loaded with 30 μL samples. The bands that were cut are outlined in green.

  • Results from gel recovery
Sample 260 280 260/280 ng/μL
pSB1A3 0.017 0.008 2.132 17.238

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

  • Use 20 ng of vector for each ligation = 1 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) 1.0 1.0 1.0
2x Roche Rapid Ligation buffer 7.0 7.0 7.0
New England Biolabs T4 ligase 1.0 1.0 1.0
dH2O 4.0 4.0 5.0
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


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