User:Karmella Haynes/Notebook/BioBrick cloning/2009/11/30

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11/30/09

  • ✓ Minipreps: KAH99(2), 100(2), 101(2), 102(2), 103(2), 104(2)
  • ✓ Assemblies: KAH011; KAH93, 94, 95 (RFP miRNA reporters) into pcDNA3.1+puro/ΔCMV (MV2)

Minipreps
> Check with E/P digests, E/P/ApaLI for KAH99, 100, 101
> ApaLI fragments = 1246, 776, 745, 497

Reagent Vol (1-6) Vol (7-12) Expected:
1,2. KAH99 = 2913
3,4. KAH100 = 3085
5,6. KAH101 = 3157
7,8. KAH102 = 374
9,10. KAH103 = 546
11,12. KAH104 = 540
miniprep digests 11/30/09
15 μL/lane; 1% agarose
DNA(plasmid) 1.0 2.0
10X buffer 1.5 1.5
EcoRI 1.0 1.0
PstI 1.0 1.0
ApaLI 1.0 ---
dH2O 9.5 9.5
  15 μL 15 μL --> 37°C/ ~15 min.

Assemblies

  1. KAH011: NLS-myc-stop (KAH011 dsOligo)/(X/P)/64 ✓ + V0120/(X/P)/3200 ✓
  2. KAH93/MV2: (1) KAH93/(X/P)/4839 + pcDNA3.1+puro/ΔCMV(MV2)/(S/P)/4529 ✓
  3. KAH94/MV2: (2) KAH94/(X/P)/5011 + "
  4. KAH95/MV2: (3) KAH95/(X/P)/5083 + "

> Digests (Fermentas FD)

Reagent Volume   Cloning digest 11/30/09
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/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH93 (X/P) 0.440 1.87 22.02
2. KAH94 (X/P) 0.165 2.97 8.24
3. KAH95 (X/P) 0.125 2.56 6.27

> Ligations

Ligation Plate results (lig : neg crtl) 12/01/09
1. KAH011(X/P)/64, 0.8 ng + V0120(X/P)/3200, 20 ng KAH011 10:1 (Pick 8)
2. V0120(X/P)/ 20 ng  
3. KAH93(X/P)/4839, 43 ng + MV2(S/P)/4529, 20 ng KAH93/MV2 1:1 (re-do)
4. KAH94(X/P)/5011, 44 ng + " KAH94/MV2 1:1 (re-do)
5. KAH95(X/P)/5083, 45 ng + " KAH95/MV2 1:1 (re-do)
6. MV2(S/P)/ 20 ng  
  1 2 3 4 5 6
Insert DNA 0.5 --- 2.0 5.3 7.0 ---
Vector DNA 4.0 4.0 0.5* 0.5* 0.5* 0.5*
2x lgn buf (Roche) 5.5 5.5 8.5 8.5 8.5 8.5
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0
dH2O --- 0.5 5.0 1.7 --- 7.0
  11μL 11μL 17μL 17μL 17μL 17μL --> R.T./~10 min; Add 25 μL Z-DH5α

--> * Dilute MV2 1:5 w/ dH2O


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