Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/21

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==Entry title==
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==11/21/12==
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* Insert content here...
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* Repeat PCR of H2B and LOV inserts
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* Note: Primers add a XbaI site upstream and the SpeI, NotI, and PstI sites downstream
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# H2B-GFP plasmid + BB_H2B fwd + BB_H2B rev
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# LOV plasmid + BB_LOV fwd + BB_LOV rev
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{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
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|-
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| Reagents || H2B || LOV
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|-
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| Plasmid DNA || 0.5 μL || 0.5
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|-
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| primer 1 (10 μM) || 1.0 || 1.0
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|-
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| primer 2 (10 μM) || 1.0 || 1.0
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|-
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| 2x GoTaq mix || 12.5 || 12.5
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|-
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| dH<sub>2</sub>O || 10.5 || 10.5
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|}
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PCR program:
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* 95°C, 3 min.
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* [95°C, 30 sec; 57°C, 30 sec.; 72°C, 30 sec.] x35 cycles
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* 72°C, 3 min.
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* 4°C ∞
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{| class="wikitable" border=0
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|-
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| <u>Expected:</u><br>1. H2B = 410 <br>2. LOV = 461
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| <br>
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|}
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<center>
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[[Image:Gel_11-9-12.png|Results of 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 PCR product digests are shown, with H2B on the left and LOV on the right. The gel was loaded with 5 μL samples.
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</center>
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Revision as of 16:54, 21 November 2012

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11/21/12

  • Repeat PCR of H2B and LOV inserts
  • Note: Primers add a XbaI site upstream and the SpeI, NotI, and PstI sites downstream
  1. H2B-GFP plasmid + BB_H2B fwd + BB_H2B rev
  2. LOV plasmid + BB_LOV fwd + BB_LOV rev
Reagents H2B LOV
Plasmid DNA 0.5 μL 0.5
primer 1 (10 μM) 1.0 1.0
primer 2 (10 μM) 1.0 1.0
2x GoTaq mix 12.5 12.5
dH2O 10.5 10.5

PCR program:

  • 95°C, 3 min.
  • [95°C, 30 sec; 57°C, 30 sec.; 72°C, 30 sec.] x35 cycles
  • 72°C, 3 min.
  • 4°C ∞
Expected:
1. H2B = 410
2. LOV = 461

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

Results of the PCR product digests are shown, with H2B on the left and LOV on the right. The gel was loaded with 5 μL samples.




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