User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/03

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01/03/10

  • Assemblies: KAH117-119/MV2; KAH94, 95/ JDS42; KAH94, 95/ JDS43



Assemblies

  1. KAH117/MV2: (1) KAH117/(E/P)/7758 + MV2/(E/P)/4529 ✓
  2. KAH118/MV2: (2) KAH118/(E/P)/7930 + "
  3. KAH119/MV2: (3) KAH119/(E/P)/8002 + "
  4. KAH94/JDS42: (4) KAH94/(E/P+Ad's)/5011 + JDS42/(BglII)/7468 ✓
  5. KAH95/JDS42: (5) KAH95/(E/P+Ad's)/5083 + "
  6. KAH94/JDS43: KAH94/(E/P+Ad's)/5011 + JDS43/(BglII)/7478 ✓
  7. KAH95/JDS43: KAH95/(E/P+Ad's)/5083 + "

> Digests (Fermentas FD)

Reagent Volume   Hover name
30 μL/lane, 1% agarose
DNA (plasmid) up to 25.0
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.

--> For KAH94 & 95: also add old purified E/P digest to melted gel/DNA for column binding.
--> Use 20 μL for final elutions.

> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH117(E/P) 0.089 6.67 4.5
2. KAH118(E/P) 0.071 3.54 3.5
3. KAH119(E/P) 0.051 4.37 2.5
4. KAH94(E/P) 0.204 2.22 10.2
5. KAH95(E/P) 0.089 1.48 4.5


> Ligations
--> Use dsoligo adapters (Ad BglII/EcoRI and Ad PstI/BglII from 12/28/09) for JDS constructs

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH117(E/P)/7758, 34 ng + MV2(E/P)/4529, 10 ng KAH117/MV2 8 colonies (Pick 4)
2. KAH118(E/P)/7930, 35 ng + " KAH118/MV2 1 colony (Pick 1)
3. KAH119(E/P)/8002, 35 ng + " KAH119/MV2 1 colony (Pick 1)
4. MV2(E/P)/ 10 ng 2 colonies
5. KAH94(E/P+Ad's)/5011, 40 ng + JDS42(BglII)/7468, 30 ng KAH94/JDS42 #:1 (Pick #)
6. KAH95(E/P+Ad's)/5083, 41 ng + " KAH95/JDS42 #:1 (Pick #)
7. JDS42(BglII)/ 30 ng  
8. KAH94(E/P+Ad's)/5011, 40 ng + JDS43(BglII)/7478, 30 ng KAH94/JDS43 #:1 (Pick #)
9. KAH95(E/P+Ad's)/5083, 41 ng + " KAH95/JDS43 #:1 (Pick #)
10. JDS43(BglII)/ 30 ng  


  1 2 3 4 5 6 7 8 9 10
Insert DNA 8.6 8.6 8.6 --- 7.1 7.1 --- 7.1 7.1 ---
Vector DNA 0.4 0.4 0.4 0.4 1.5 1.5 1.5 1.5 1.5 1.5
Ad BglII/EcoRI --- --- --- --- 0.2 0.2 0.2 0.2 0.2 0.2
Ad PstI/BglII --- --- --- --- 0.2 0.2 0.2 0.2 0.2 0.2
2x lgn buf (Roche) 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O --- --- --- 8.6 --- --- 7.1 --- --- 7.1
  20μL 20μL 20μL 20μL 20μL 20μL 20μL 20μL 20μL 20μL

--> R.T./ 30 min.; Add 30 μL Z-DH5α



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