Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/06

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November 6, 2012

  • Restriction digests of H2B-GFP and LOV
Restriction Digest Reagents
Reagent H2B-GFP/EcoRI H2B-GFP/PstI LOV/PstI
DNA(plasmid) 2.0 2.0 2.0
10x buffer 1.5 1.5 1.5
EcoRI 1.0 0 0
PstI 0 1.0 1.0
dH2O 10.5 10.5 10.5
Total 15.0 15.0 15.0


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

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


  • ---Karmella 16:01, 7 November 2012 (EST): Note: Please re-do LOV/PstI


  • ---Karmella 16:01, 7 November 2012 (EST): Here is a reaction table for PCR
  • First, resuspend dry primer oligos (in blue-capped tubes) to 100 μM
  • Then make working solutions of 10 μM
  • Reactions:
  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 ∞


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