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
  1. H2B

1A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG

H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA

  1. 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

Results of the digests are shown. KAH Fermentas GeneRuler 1kbplus.jpg

Results of the PCR product digests are shown. It was later found that the transparency used was blocking the UV light, so no bands could be seen.

  • 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 0.078 0.043 1.81 77.995
LOV 0.08 0.044 1.817 80.443
pSB1A3 0.023 0.014 1.727 23.386