User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/22

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12/22/09

  • ✓ Cultures: KAH120-125 in MV2 (2 each); KAH135 (2)
  • ✓ Digests/ dephos.: Vectors for Strept apt into JDS42 & 43
  • ✓ Kinase treatment + oligo annealing: Strept apt

Assemblies

  1. Strep apt/JDS42: Strept apt/(NotI dsoligo)/50 + (1) JDS42/(NotI)/7468
  2. Strep apt/JDS43: Strept apt/(NotI dsoligo)/50 + (2) JDS43/(NotI)/7478

> Digests (Fermentas FD)

Reagent Volume   Cloning digest 12/22/09
30 μL/lane, 1% agarose
DNA (plasmid) 10.0
10x buffer 3.0
NotI 2.0
dH2O 15.0
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. JDS42(NotI) 0.053 2.08 5.6
2. JDS43(NotI) 0.139 3.24 6.9


> Dephosphorylation (Roche)

Reagent Volume
DNA (clean digest) up to 17 μL (~100 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~5 ng/μL

Kinase treatment & oligo annealing

  1. Joe's: Strep apt S1 F NotI + Strep apt S1 R NotI

> Kinase treatment

DNA (oligos, 100 μM) up to 23 μL (3 μL ea.)
10 x PNK (polynucleotide kinase) buffer 3.0
10mM ATP 2.0
T4 polynucleotide kinase (PNK) 2.0
dH2O ---
  30 μL

--> 37°C/ 1.5 hours
--> Add 4 uL 500 mM NaCl
--> 100°C (water bath)/ 5 min.; Cool to R.T. overnight in water bath


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