User:Karmella Haynes/Notebook/BioBrick cloning/2010/02/19

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02/19/10

  • ✓ DPRE cloning: BB PCR
  • ✓ Assmebly: DPRE into V0120

PCR
--> Template: DPRE from CJW
--> Primers: BBp_DPRE/ BBs_DPRE

Reagent Volume
DNA(plasmid) 0.5
10 μM primer mix 1.0
2x GoTaq Green 15
dH2O 13.5
  30 μL

--> BioRad Block A

  • 95°C/ 3 min.
  • [95°C/ 1 min., 57°C/ 1 min., 72°C/ 2 min.] x30
  • 72°C/ 3 min.
  • 4°C/ ∞

--> Zymo clean & concentrate; elute w/ 20 μL dH2O



Assembly

  • Note: DPRE is not a standard BioBrick. Will need to mutate 2 PstI sites to make it a BioBrick. For preliminary in vivo tests, the wild-type form will be used (without PstI cloning)
  1. DPRE: (1) DPRE PCR/(E/S)/1821 + (2) V0120/(E/S)/3200


> Digests

Reagent 1 2 Expected:
1. DPRE = 1821
File:KAH 021910 gel1.tif
30 μL/ lane; 1% agarose
DNA(clean PCR) 20.0 15.0
10x buffer 3.0 3.0
EcoRI 1.0 1.0
SpeI 1.0 1.0
dH2O 5.0 10.0
  30 30 μL --> 37°C/ 30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. DPRE PCR (X/P) 0.839 1.78 41.9
2. EF-1α PCR 2(X/P) 0.155 1.49 7.7


> Ligations

Ligation Plate results (lig : neg crtl) 02/22/10
1. DPRE PCR(E/S)/1821, 23 ng + V0120(E/S)/3200, 20 ng DPRE >10:1 (Pick 4)
2. V0120(E/S)/ 20 ng
  1 2
Insert DNA 0.5 ---
Vector DNA 2.5 2.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 1.0 1.5
  10μL 10μL --> R.T./ ~10 min.; Add 25 μL z-DH5α