User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/10

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12/10/09

  • ✓ Minipreps: KAH109-116 (2 each, 16 total)
  • ✓ Lab meeting at 4 pm! (Kevin and Karmella)
  • ✓ Assemblies: KAH126-133; KAH134
  • ✓ Cultures: KAH120-125 (3 each, 18 total)

Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. KAH109 = 1123
3,4. KAH110 = 1123
5,6. KAH111 = 1123
7,8. KAH112 = 1123
9,10. KAH113 = 2107
11,12. KAH114 = 2110
13,14. KAH115 = 931
15,16. KAH116 = 775
File:KAH 121009 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~15 min.


Assemblies

  1. KAH126: CMVTetO2-K(KAH3)/(S/P)/3951 ✓ + (1) KAH109/(X/P)/1123
  2. KAH127: " + (2) KAH110/(X/P)/1123
  3. KAH128: " + (3) KAH111/(X/P)/1123
  4. KAH129: " + (4) KAH112/(X/P)/1123
  5. KAH130: " + (5) KAH113/(X/P)/2107
  6. KAH131: " + (6) KAH114/(X/P)/2110
  7. KAH132: " + (7) KAH115/(X/P)/931
  8. KAH133: " + (8) KAH116/(X/P)/775
  9. KAH134: (10) Streptavidin tag(Joe)/(S/P)~3200 + (9) KAH67/(X/P)/1353

> Digests (Fermentas FD)

Reagent Volume   File:KAH 121009 gel2.tif
30 μL/lane, 1% agarose
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~1 hr.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH109(X/P) 0.490 1.97 24.5
2. KAH110(X/P) 0.444 2.06 22.2
3. KAH111(X/P) 0.463 1.87 23.2
4. KAH112(X/P) 0.474 1.94 23.7
5. KAH113(X/P) 0.816 2.03 40.8
6. KAH114(X/P) 0.748 2.11 37.4
7. KAH115(X/P) 0.322 2.28 16.1
8. KAH116(X/P) 0.295 2.28 14.7
9. KAH67(X/P) 0.794 1.93 39.7
10. Strep(X/P) 0.736 1.91 36.8

> Ligations

Ligation Plate results (lig : neg crtl) 12/11/09
1. KAH109(X/P)/1123, 11 ng + KAH03(S/P)/3951, 20 ng KAH126 >10:1 (Pick 2)
2. KAH110(X/P)/1123, 11 ng + " KAH127 >10:1 (Pick 2)
3. KAH111(X/P)/1123, 11 ng + " KAH128 >10:1 (Pick 2)
4. KAH112(X/P)/1123, 11 ng + " KAH129 >10:1 (Pick 2)
5. KAH113(X/P)/2107, 21 ng + " KAH130 >10:1 (Pick 2)
6. KAH114(X/P)/2110, 21 ng + " KAH131 >10:1 (Pick 2)
7. KAH115(X/P)/931, 9 ng + " KAH132 >10:1 (Pick 2)
8. KAH116(X/P)/775, 8 ng + " KAH133 >10:1 (Pick 2)
9. KAH03(S/P)/ 20 ng  
10. KAH67(X/P)/1353, 17 ng + Strep(S/P)/3200, 20 ng KAH134 >10:1 (Pick 2)
11. Strep(S/P)/ 20 ng  
  1 2 3 4 5 6 7 8 9 10 11
Insert DNA 0.5 0.5 0.5 0.5 0.6 0.6 0.6 0.6 --- 0.5 ---
Vector DNA 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.5 0.5
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 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 1.0 1.0
dH2O 2.8 2.8 2.8 2.8 2.7 2.7 2.7 2.7 3.3 3.0 3.5
  10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL --> R.T./ >10 min.; Add 25 μL Z-DH5α