Haynes Lab:Notebook/SynBERC Scholars 2013/2014/03/17

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03/17/14

  • Luc 14 genomic DNA test - restriction digest and gel
  • Luc 14 genomic DNA clean-up & PCR



Luc 14 genomic DNA test - restriction digest and gel

  • Check genomic DNA on a gel, with or without restriction digestion
  1. Luc 14 HEK - No enzyme
  2. Luc 14 HEK - EcoRI (fast digest)
  3. Luc 14 HEK - PstI (fast digest)
  4. U2OS plain - No enzyme
  5. U2OS plain - EcoRI (fast digest)
  6. U2OS plain - PstI (fast digest)
REAGENT 1 2 3 4 5 6 Inconclusive: Gel showed no DNA smear
Genomic DNA (~500 ng) 2.0 μL 2.0 2.0 1.0 1.0 1.0
Rest. enzyme 0 2.0 2.0 0 2.0 2.0
10x buffer 2.0 2.0 2.0 2.0 2.0 2.0
dH2O 16.0 14.0 14.0 17.0 15.0 15.0
  20.0 20.0 20.0 20.0 20.0 20.0


Luc 14 genomic DNA clean-up & PCR

  • Clean up 1/2 of the Luc genomic DNA (50 μL) on a Zymo clean and concentrator column
    • 50 μL ~ 4340 ng
    • Eluted with 20 μL dH2O ~ 217 ng/μL
  1. GAPDH A3 - #359, 360
  2. GAPDH B2 - #361, 362
  3. MMP12 A3 - #325, 326
  4. MMP12 B2 - #367, 368
  5. MMP12 C2 - #327, 328
  6. luc 1 - luc f1, luc r1 - #724, 725
  7. luc 2 - luc f2, luc r2 - #726, 727
  8. luc 4 - luc f4, luc r4 - #730, 731
  9. Kozak plasmid + P0001/P0002
  10. Kozak plasmid + P0001/P0002 (again)


Reagent Kozak Luc14 EXPECTED
1. GAPDH A3 = 125 bp
2. GAPDH B2 = 112
3. MMP12 A3 = 100
4. MMP12 B2 = 118
5. MMP12 C2 = 103
6. luc 1 = 110
7. luc 2 = 98
8. luc 4 = 105
9, 10. Kozak = 277
Primer check gel
2% agarose gel, 20 μL/lane, 1kb+ Ladder
DNA 0.5 μL 0.5
P0001 1.0 ---
P0002 1.0 ---
mixed primers --- 1.0
2x GoTaq 12.5 12.5
dH2O 10.0 11.0
  25.0 25.0

PCR

  • 95°C, 60 sec
  • [95°C, 15 sec; 57°C, 15 sec; 72°C 15 sec] x35
  • 72°C, 60 sec
  • 4°C ∞