Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/02/08

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February 8, 2013

Type IIS Assembly - Making PCR Products

  • Attempt new assembly method because piecewise assembly was unsuccessful
  • Final assembly product: pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
  • Primer design

H2B 1A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA

LOV LOV fwd 5'-cacaccaCGTCTCa TTGGCTACTACACTT LOV+his/1A3 rev 5'-cacaccaCGTCTCa TAGT ttagtggtgatggtgatgatgAAGTTCTTTTGCCGC

  • First, resuspend dry primer oligos (in blue-capped tubes) to 100 μM
  • Then make working solutions of 10 μM
  • Reactions:
  1. H2B-GFP plasmid + 1A3/H2B fwd + H2B/LOV rev
  2. LOV plasmid + LOV fwd + LOV+his/1A3 rev
Reagents H2B LOV
Plasmid DNA 0.2 μL 0.2
primer 1 (10 μM) 1.0 1.0
primer 2 (10 μM) 1.0 1.0
2x GoTaq mix 25.0 25.0
dH2O 22.8 22.8
Total 50.0 50.0

PCR program:

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

Image:Gel 2-8-13.png 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.

  • The PCR products were purified with the Zymo DNA Clean & Concentrator Kit and eluted with 20 μL dH2O
PCR Product 260 280 260/280 ng/μL
H2B fill fill fill fill
LOV fill fill fill fill



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