User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/25

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01/25/10

  • ✓ Colony PCR screen: KAH 94, 95/ JDS48, 49; HS4 primers
  • ✓ Assembly: KAH140, 141/ V0201 (re-do, d.p. vector)
  • ✓ Cultures: KAH94/JDS48 #1 & KAH94/JDS49 #18 (2 each)

Colony PCR screen
> Use BB_HS4 f1 & BB_HS4 r1 primers
> Controls: (+) = KAH94 (0.5μL); (-) = JDS48 (0.5μL)

Reagent Volume Expected:
340, 686 bp
Colony PCR 1/25/10
15 μL/lane; 1% agarose
E. coli from streak 0.0
10μM primer mix 1.0
2x GoTaq Green 10.0
dH2O 9.0
  20 μL
 
 
 
 
 

--> BioRad 96-well:

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x30
  • 72°C/ 3 min.
  • 4°C/ ∞

--> Check minipreps of KAH94/JDS48 #1 & KAH94/JDS49 #18 tomorrow with BglII and PstI digests


Assemblies

  1. KAH140/V0201: KAH140/(N/S)/2945 + V0201/(N/S, d.p.)/5157
  2. KAH141/V0201: KAH141/(N/S)/2939 + "


> Dephosphorylation (Roche)
--> d.p. treat V0201(N/S)

Reagent Volume
DNA (clean digest) 17.0 (~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

Ligation Plate results (lig : neg crtl) 01/26/10
1. KAH140(N/S)/2939, 29 ng + V0201(N/S)/5127, 25 ng KAH140/V0201 >10:1 (Pick 2)
2. KAH141(N/S)/2527, 25 ng + " KAH141/V0201 >10:1 (Pick 2)
3. V0201(N/S)/ 25 ng  
  1 2 3
Insert DNA 3.0 3.0 ---
Vector DNA 1.0 1.0 1.0
2x lgn buf (Roche) 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0
dH2O --- --- 3.0
  10μL 10μL 10μL --> R.T./ 10 min.; Add 20 μL Z-DH5α



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