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

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==Entry title==
==11/21/12==
* Insert content here...
 
* Repeat PCR of H2B and LOV inserts
* Note: Primers add a XbaI site upstream and the SpeI, NotI, and PstI sites downstream
 
# H2B-GFP plasmid + BB_H2B fwd + BB_H2B rev
# LOV plasmid + BB_LOV fwd + BB_LOV rev
 
{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
|-
| 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
|-
| dH<sub>2</sub>O || 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 ∞
 
{| class="wikitable" border=0
|-
| <u>Expected:</u><br>1. H2B = 410 <br>2. LOV = 461
| <br>
|}
 
<center>
[[Image:Gel_11-9-12.png|Results of the digests are shown.]]
[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]
</center>
 
<center>
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.
</center>
 
 




Revision as of 13:54, 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.

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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