User:Karmella Haynes/Notebook/BioBrick cloning/2012/06/17

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06/17/12

  • PCR clone: Lac promoter from Francesca's GFP switch (P2547 O2)



Assembly

  1. PlacO2: P2547-O2/(X/P)/~100 + V0120/(X/P)/3200

> PCR

  • Use primers P2547 O2 F/ P2547 O2 R
  • Use O2 GFP as template
Reagent Vol
DNA plasmid 0.4 ul
10 uM fwd primer 1.0
10 uM rev primer 1.0
2x Go Taq 25.0
dH2O 22.6
  50 ul

--> Set up 2 reactions
--> Wang lab BioRad PCR

  • 95C/ 3 min
  • (95C/ 30 sec, 55C/ 30 sec, 72C/ 30 sec) x35
  • 72C/ 3 min
  • 4C/ forever


> Zymo clean PCR
--> Elute each sample with 25 ul dH2O


> Digests (Fermentas FD)
1, 2. Digest entire 25 ul for the PCR samples
3. Use KAH160 to get V0120 backbone (10 ul plasmid)

Reagent Volume   Cloning digest 6/17/12
30 μL/lane, 1% agarose, GeneRuler ladder
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.

--> Combine bands 1, 2
--> Zymo gel DNA recovery, elute with 15 ul


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. P2547 O2 (X/P) 0.009 1.75 9.465
2. V0120 (X/P) 0.002 2.125 1.765

--> Very low yield for V0120 vector (13.0 ul total). Maximum possible amount per ligation = 11 ng (6.5 ul)


> Ligations

Ligation Plate results (lig : neg crtl) 06/18/12
1. P2547 O2 (X/P)/~100, 0.68 ng + V0120 (X/P)/3200, 11 ng PlacO2 no colonies
2. V0120 (X/P)/ 11 ng  
  1 2
Insert DNA 0.5 ---
Vector DNA 6.5 6.5
2x lgn buf (Roche) 8.0 8.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 0.5
  16.0 μL 16.0 μL

--> Add 40 ul DH5α Turbo; spread on 100 ug/ml Amp