User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/16

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12/16/09

  • ✓ Digest: confirm KAH105-108 (& /MV2) via E/P digests
  • ✓ Minipreps: KAH126-133/MV2 (2 ea); KAH123, 125 (6 ea)
  • ✓ Assemblies: KAH117-119; KAH135, 136

Digest check
> May have mixed up and mislabeled KAH105-108 (& /MV2). Sequencing results are out of order (see | 12/09/09). Could be cause of last failed transfection experiment.

Reagent Volume Expected:
MV2 = 4529
1. KAH105 = 2794
2. KAH105/MV2 = 2794
3. KAH106 = 2788
4. KAH106/MV2 = 2788
5. KAH107 = 2966
6. KAH107/MV2 = 2966
7. KAH108 = 2960
8. KAH108/MV2 = 2960
K-stop testers, check 12/16/09
15 μL/lane; 1% agarose
DNA(plasmid) 1.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 10.5
  15 μL --> 37°C/ ~15 min.

Results are consistent with the sequencing data: 1 = KAH108, 3 = KAH105, 5 = KAH106, 7 = KAH107
--> Correct labeling on the tubes


Minipreps
> Check with E/P digests

Reagent Volume Expected:
MV2 = 4529
1,2. KAH126/MV2 = 1880
3,4. KAH127/MV2 = 1880
5,6. KAH128/MV2 = 1880
7,8. KAH129/MV2 = 1880
9,10. KAH130/MV2 = 2864
11,12. KAH131/MV2 = 2867
13,14. KAH132/MV2 = 1688
15,16. KAH133/MV2 = 1532
17-22. KAH123 = 8002
23-28. KAH125 = 8246
E/P digests 12/16/09
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. KAH117: KAH99/(E/S)/2913 ✓ + (1) KAH93/(E/X)/8039
  2. KAH118: KAH100/(E/S)/3085 ✓ + "
  3. KAH119: KAH101/(E/S)/3157 ✓ + "
  4. KAH135: KAH78/(E/S)/3652 ✓ + (2) KAH134/(E/X)/4600
  5. KAH136: KAH78/(E/S)/3652 ✓ + "

> Digests (Fermentas FD)

Reagent Volume   Cloning digests 12/16/09
30 μL/lane, 1% agarose
DNA (plasmid) 10.0
10x buffer 3.0
enzyme 1 2.0
enzyme 2 2.0
dH2O 13.0
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH93(E/X) 0.283 2.49 14.2
2. KAH134(E/X) 0.367 1.94 18.3


> Dephosphorylation (Roche)

Reagent Volume
DNA (clean digest) up to 17 μL (250 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 12.5 ng/μL


> Ligations

Ligation Plate results (lig : neg ctrl) 12/17/09
1. KAH99(E/S)/2913, 29 ng + KAH93(E/X)/8039, 40 ng KAH117 too few col's (re-do)
2. KAH100(E/S)/3085, 31 ng + " KAH118 too few col's (re-do)
3. KAH101(E/S)/3157, 31 ng + " KAH118 too few col's (re-do)
4. KAH93(E/X)/ 40 ng  
5. KAH78(E/S)/3652, 41 ng + KAH134(E/X)/4600, 20 ng KAH135 too few col's (re-do)
6. KAH79(E/S)/3724, 41 ng + " KAH136 too few col's (re-do)
7. KAH134(E/X)/ 20 ng  
  1 2 3 4 5 6 7
Insert DNA 0.5 0.6 0.8 --- 2.0 2.0 ---
Vector DNA 3.2 3.2 3.2 3.2 1.6 1.6 1.6
2x lgn buf (Roche) 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
dH2O 0.3 0.2 --- 0.8 0.4 0.4 2.4
  10μL 10μL 10μL 10μL 10μL 10μL 10μL --> R.T./ ~15 min.; Add 25 μL Z-DH5α



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