Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/02

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November 2, 2012 Digestion and PCR Planning

  • Check LOV and H2B-GFP minipreps with digestion
  • BioBrick PCR

Minipreps

Measure concentrations

Sample 260/280 ng/μL
LOV 1.834 281.868
H2B-GFP 1.87 118.217

Check miniprep results

  • Use a plasmid editor software to check sequences for restriction digest enzymes
  • LOV has two PstI sites while H2B-GFP has one PstI, one NotI, and two EcoRI sites
  • Decide to cut LOV with PstI and H2B-GFP with PstI and EcoRI
  • Expected bands for LOV at 251 with PstI digest
  • Expected bands for H2B-GFP at 4885, 205 with Psti and EcoRI digest


Reagent LOV H2B-GFP/ PstI H2B-GFP/ EcoRI
DNA(plasmid) 2.0 μL 2.0 2.0
10X buffer 1.5 1.5 1.5
PstI 1.0 1.0 ---
EcoRI --- --- 1.0
dH2O 10.5 10.5 10.5
  15.0 15.0 15.0

Results

Expected:
1. LOV/PstI = 251
2. H2B-GFP/ PstI = 5101
3. H2B-GFP/ EcoRI = 4885, 210

15 μL/lane; 1% agarose



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