User:Karmella Haynes/Notebook/BioBrick cloning/2012/06/27

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06/27/12

  • ✓ Assemblies: PLinkers into V0120



Assemblies

  1. PLrigid/V0120: PLrigid/(X/P)/60 + V0120/(X/P)/~3200
  2. PLrigid4/V0120: PLrigid4/(X/P)/240 + "
  3. PLflex/V0120: PLflex/(X/P)/60 + "
  4. PLflex4/V0120: PLflex4/(X/P)/240 + "


> Digests (Fermentas FD)
--> Will use dephos. methods for these small inserts

Reagent Volume  
DNA (plasmid) 20.0
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 5.0
  30 μL --> 37°C/ ~30 min.

---> Zymo clean; elute w/ 15 μL dH2O


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. PLrigid (X/P) 0.036 1.842 36.1
2. PLrigid4 (X/P) 0.038 1.851 38.4
3. PLflex (X/P) 0.036 1.839 36.2
4. PLflex4 (X/P) 0.054 1.841 53.7


> Dephosphorylation (Roche)

Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


> Ligations
--> 60 bp inserts = 1/50 total mass; 240 bp inserts are 1/12 of total mass; multiply ng used accordingly
--> Use V0120 from PEST

Ligation Plate results (lig : neg crtl) 06/28/12
1. PLrigid(X/P)/60, 1 ng (x50) + V0120(X/P)/3200, 25 ng PLrigid/V0120 10:1 (Pick 2)
2. PLrigid4(X/P)/240, 4 ng (x12) + " PLrigid4/V0120 10:1 (Pick 2)
3. PLflex(X/P)/60, 1 ng (x50) + " PLflex/V0120 10:1 (Pick 2)
4. PLflex4(X/P)/240, 4 ng (x12) + " PLrigid4/V0120 10:1 (Pick 2)
2. V0120(X/P)/ 25 ng  
  1 2
Insert DNA 2.0 2.0 2.0 2.0 ---
Vector DNA 0.5 0.5 0.5 0.5 0.5
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0
dH2O 1.5 1.5 1.5 1.5 3.5
  10 μL 10 μL 10 μL 10 μL 10 μL

--> Add 30 μL DH5α Turbo cells; plate on 100 μg/mL Amp