User:Karmella Haynes/Notebook/BioBrick cloning/2012/12/11

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Karmella's BioBrick Cloning Main project page
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12/11/12

  • Note: colonies from retransformation of pSB1A3-RFP look red
  • Assembly: KAH111 + C-His (trouble shooting for Behzad, start from plasmids)
  • Minipreps: Rene's pSB1A3-RFP (2); cut with X/S for Brady and Vi's Gibson cloning


Assemblies

  1. KAH111/C-His: 5' part/(E/P)/1123 + C-His/(E/P)/3600


  • Digests (Fermentas FD)
Reagent Volume Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 15.0 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 10.0
  30 μL --> 37°C/ ~30 min.


  • Measure conc.'s of DNA purified from gel (Zymo kit)
Sample OD260 260/280 ng/μL
1. KAH11 (E/P) 0.019 1.96 18.7
2. C-His (E/P) 0.047 1.82 46.8


  • Ligations
Ligation Plate results (lig : neg crtl) 12/12/12
1. KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 25ng KAH111/C-His 3:1 (Pick 4) (very small colonies after ~18 hours)
2. C-His(E/P)/ 25 ng 3 colonies
  1 2
Insert DNA 1.0 ---
Vector DNA 0.5 0.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 2.5 3.5
  10 μL 10 μL


Minipreps

  • pSB1A3-RFP (retransformation from 12/10/12)
  • Note: pellets are red
  • Digest 10 μL of each with X/S for Gibson cloning (Brady and Vi)
  • Note: combined mini preps afterwards; concentration = 60 ng/μL
Reagent Volume Expected:
3. pSB1A3 = 2155
4. pSB1A3 = 2155
 Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 10.0 μL
10X buffer 3.0
XbaI 1.0
SpeI 1.0
dH2O 15.0
  15 μL --> 37°C/ ~30 min.


  • Gel purification: combined the two slices
    • Concentration: 260 = 0.015, 260/280 = 2.2, ng/μL = 15.1



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