Difference between revisions of "Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/12"

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==November 12, 2012==
 
==November 12, 2012==
−
* 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
 
|-
 
|-
−
| LOV 0.048 || 0.026 || 1.86 || 48.095
+
| 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 [http://partsregistry.org/Part:pSB1A2 pSB1A2 plasmid] (from Rene) XbaI and SpeI
 +
 +
{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
 +
|-
 +
| 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
 +
|-
 +
| dH<sub>2</sub>O || --- || --- || ---
 +
|-
 +
| 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
  
  

Revision as of 05:51, 15 November 2012

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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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