User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/14

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12/14/09

  • ✓ Minipreps: digests for 12/12/09 minipreps
  • ✓ Cultures: KAH123(3), 124(3), 125(3)
  • ✓ Assemblies: KAH126-133 into MV2

Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. KAH126 = 1880
3,4. KAH127 = 1880
5,6. KAH128 = 1880
7,8. KAH129 = 1880
9,10. KAH130 = 2864
11,12. KAH131 = 2867
13,14. KAH132 = 1688
15,16. KAH133 = 1532
17,18. KAH134 = 1400
File:KAH 121409 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/MV2: (1) KAH126/(E/P)/1880 + MV2/(E/P)/4529 ✓
  2. KAH127/MV2: (2) KAH127/(E/P)/1880 + "
  3. KAH128/MV2: (3) KAH128/(E/P)/1880 + "
  4. KAH129/MV2: (4) KAH129/(E/P)/1880 + "
  5. KAH130/MV2: (5) KAH130/(E/P)/2864 + "
  6. KAH131/MV2: (6) KAH131/(E/P)/2867 + "
  7. KAH132/MV2: (7) KAH132/(E/P)/1688 + "
  8. KAH133/MV2: (8) KAH133/(E/P)/1532 + "

> Digests (Fermentas FD)
--> Triple digest KAH130 & 131 w/ E/P/ScaI

Reagent Vol (1-4, 7, 8) Vol (5, 6) File:KAH 121409 gel2.tif
30 μL/lane, 1% agarose
DNA (plasmid) 15.0 15.0
10x buffer 3.0 3.0
enzyme 1 1.0 1.0
enzyme 2 1.0 1.0
ScaI --- 1.0
dH2O 10.0 9.0
  30 μL 30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH126(E/P) 0.705 1.86 35.2
2. KAH127(E/P) 1.095 1.88 54.7
3. KAH128(E/P) 0.916 1.88 45.8
4. KAH129(E/P) 0.892 1.85 44.6
5. KAH130(E/P) 0.776 2.02 38.8
6. KAH131(E/P) 0.620 1.93 31.0
7. KAH132(E/P) 0.729 1.88 36.5
8. KAH133(E/P) 0.380 1.96 19.0

> Ligations

Ligation Plate results (lig : neg crtl) 12/15/09
1. KAH126(E/P)/1880, 21 ng + MV2/(E/P)/4529, 25 ng KAH126/MV2 10:1 (Pick 2)
2. KAH127(E/P)/1880, 21 ng + " KAH127/MV2 10:1 (Pick 2)
3. KAH128(E/P)/1880, 21 ng + " KAH128/MV2 10:1 (Pick 2)
4. KAH129(E/P)/1880, 21 ng + " KAH129/MV2 10:1 (Pick 2)
5. KAH130(E/P)/2864, 32 ng + " KAH130/MV2 10:1 (Pick 2)
6. KAH131(E/P)/2867, 32 ng + " KAH131/MV2 10:1 (Pick 2)
7. KAH132(E/P)/1688, 19 ng + " KAH132/MV2 10:1 (Pick 2)
8. KAH133(E/P)/1532, 17 ng + " KAH133/MV2 10:1 (Pick 2)
2. MV2(E/P)/ 25 ng  
  1 2 3 4 5 6 7 8 9
Insert DNA 1.0 1.0 1.0 1.0 1.5 1.5 1.0 1.0 ---
Vector DNA 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
2x lgn buf (Roche) 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
dH2O 2.0 2.0 2.0 2.0 1.5 1.5 2.0 2.0 3.0
  10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL --> R.T./ ~10 min.; Add 20μL Z-DH5α