Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/12

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(November 12, 2012)
Current revision (07:51, 15 November 2012) (view source)
(November 12, 2012)
 
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==November 12, 2012==
==November 12, 2012==
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* The PCR products were purified with the Zymo DNA Clean and Concentration Kit
+
* The PCR products were purified with the Zymo DNA Clean & Concentrator Kit
{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
Line 15: Line 15:
| H2B || 0.052 || 0.028 || 1.865 || 51.607
| H2B || 0.052 || 0.028 || 1.865 || 51.607
|-
|-
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| LOV || 0.048 || 0.026 || 1.86 || 48.095
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| LOV || 0.048 || 0.026 || 1.860 || 48.095
|}
|}
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 +
 +
----
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Great! Next step...
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 +
* Digests:
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** In a 30 μL reaction, cut H2B with XbaI and SpeI
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** In a 30 μL reaction, cut LOV with XbaI and SpeI
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** In a 30 μL reaction, cut a [http://partsregistry.org/Part:pSB1A2 pSB1A2 plasmid] (from Rene) XbaI and SpeI
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 +
{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
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|-
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| Reagent || H2B|| LOV || pSB1A2
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|-
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| DNA || 8.0 || 8.0 || 15.0
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|-
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| XbaI || 1.0|| 1.0 || 1.0
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|-
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| SpeI || 1.0 || 1.0 || 1.0
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|-
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| 10x buffer || 3.0 || 3.0 || 3.0
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|-
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| dH<sub>2</sub>O || --- || --- || ---
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|-
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| Total vol. || 30. 0 || 30.0  || 30.0
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|}
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* Run entire 30 μL on a 1% gel (use the big fat-tooth well comb)
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* Cut band out of gel (use UV box); H2B = ~410, LOV = ~461, pSB1A2 = ~2000
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* Extract DNA from gel

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November 12, 2012

  • The PCR products were purified with the Zymo DNA Clean & Concentrator Kit
PCR Product 260 280 260/280 ng/μL
H2B 0.052 0.028 1.865 51.607
LOV 0.048 0.026 1.860 48.095



Great! Next step...

  • Digests:
    • In a 30 μL reaction, cut H2B with XbaI and SpeI
    • In a 30 μL reaction, cut LOV with XbaI and SpeI
    • In a 30 μL reaction, cut a pSB1A2 plasmid (from Rene) XbaI and SpeI
Reagent H2B LOV pSB1A2
DNA 8.0 8.0 15.0
XbaI 1.0 1.0 1.0
SpeI 1.0 1.0 1.0
10x buffer 3.0 3.0 3.0
dH2O --- --- ---
Total vol. 30. 0 30.0 30.0


  • Run entire 30 μL on a 1% gel (use the big fat-tooth well comb)
  • Cut band out of gel (use UV box); H2B = ~410, LOV = ~461, pSB1A2 = ~2000
  • Extract DNA from gel



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