User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/21

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12/21/09

  • ✓ Assemblies: KAH120-125 in MV2; KAH135
  • ✓ Oligo annealing: Joe's "Strept apt" tag (Forgot kinase treatment, do tomorrow)

Assemblies

  1. KAH120/MV2: (1) KAH120/(E/P)/7930 + MV2/(E/P)/4529 ✓
  2. KAH121/MV2: (2) KAH121/(E/P)/8102 + "
  3. KAH122/MV2: (3) KAH121/(E/P)/8174 + "
  4. KAH123/MV2: (4) KAH121/(E/P)/8002 + "
  5. KAH124/MV2: (5) KAH121/(E/P)/8174 + "
  6. KAH125/MV2: (6) KAH121/(E/P)/8246 + "
  7. KAH135: (7) Gal4DB-PEST(KAH55)/(S/P)/4146 + EED/(X/P)/1401 ✓

> Digests (Fermentas FD)

Reagent Volume   File:KAH 122109 gel1.tif
30 μL/lane, 1% agarose
DNA (plasmid) 10.0
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 15
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH120(E/P) 0.096 5.00 4.8
2. KAH121(E/P) 0.113 2.01 5.7
3. KAH122(E/P) 0.150 2.46 7.5
4. KAH123(E/P) 0.208 1.66 10.4
5. KAH124(E/P) 0.074 3.63 3.7
6. KAH125(E/P) 0.198 1.94 9.9
7. KAH55(S/P) 0.792 1.96 39.6

> Ligations

Ligation Plate results (lig : neg crtl) 12/22/09
1. KAH120(E/P)/7930, 35 ng + MV2(E/P)/4529, 10 ng KAH120/MV2 8 colonies (Pick 2)
2. KAH121(E/P)/8102, 36 ng + " KAH121/MV2 9 colonies (Pick 2)
3. KAH122(E/P)/8174, 36 ng + " KAH122/MV2 10 colonies (Pick 2)
4. KAH123(E/P)/8002, 35 ng + " KAH123/MV2 6 colonies (Pick 2)
5. KAH124(E/P)/8174, 36 ng + " KAH124/MV2 5 colonies (Pick 2)
6. KAH125(E/P)/8246, 36 ng + " KAH125/MV2 7 colonies (Pick 2)
7. MV2(E/P)/ 10 ng 0 colonies
8. EED(X/P)/1401, 14 ng + KAH55(S/P)/4146, 20 ng KAH135 >10:1 (Pick 2)
9. KAH55(S/P)/ 20 ng  
  1 2 3 4 5 6 7 8 9
Insert DNA 8.6 8.6 8.6 8.6 8.6 8.6 --- 0.5 ---
Vector DNA 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.5 0.5
2x lgn buf (Roche) 10.0 10.0 10.0 10.0 10.0 10.0 10.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O --- --- --- --- --- --- 8.6 3.0 3.5
  20μL 20μL 20μL 20μL 20μL 20μL 20μL 10μL 10μL ---> R.T./ 1 hr.; Add 30 μL (1-7) or 20 μL (8, 9) Z-DH5α

Oligo annealing

  1. Strept apt (Joe's oligos, NotI sticky ends)
DNA (oligos, 100 μM) up to 18 μL (3 μL ea.)
10x annealing buffer 2.0
dH2O ---
  20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight