User:Karmella Haynes/Notebook/BioBrick cloning/2010/04/09

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04/09/10

  • ✓ Minipreps: KAH142 rebuilt (4)
  • ✓ Assemblies: KAH146, 147 (H3me3 reporters) and KAH148-153 (Pc-ATF's w/o VP64, controls)



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1-4. KAH142 = 4571
File:KAH 040910 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. KAH146: (1) KAH96/(E/S/ApaL1)/2825 + (3) KAH145/(E/X)/~6755
  2. KAH147: (2) KAH142/(E/S)/4571 + "
  3. KAH148: KAH01/(E/S dp)/186 + (4) KAH116/(E/X)/~3975
  4. KAH149: KAH02/(E/S dp)/186 + "
  5. KAH150: KAH03/(E/S dp)/186 + "
  6. KAH151: KAH04/(E/S dp)/186 + "
  7. KAH152: KAH06/(E/S)/1170 + "
  8. KAH153: KAH07/(E/S)/1173 + "

> Digests (Fermentas FD)
--> Triple digest KAH96 w/ E/S/ApaLI

Reagent Volume   File:KAH 040910 gel2.tif
30 μL/lane, 1% agarose
DNA (plasmid) up to 25 μL up to 24 μL
10x buffer 3.0 3.0
enzyme 1 1.0 1.0
enzyme 2 1.0 1.0
ApaLI --- 1.0
dH2O --- ---
  30 μL 30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH96 (E/S/ApaLI) 0.393 1.75 19.6
2. KAH142 (E/S) 0.394 1.91 19.7
3. KAH145 (E/X) 0.371 1.71 18.6
4. KAH116 (E/X) 0.762 1.85 38.1


> Ligations

Ligation Plate results (lig : neg crtl) 04/10/10
1. KAH96(E/S)/2825, 33 ng + KAH145(E/X)/6755, 40 ng KAH146 1:1 (re-do)
2. KAH142(E/S)/4571, 54 ng + " KAH147 1:1 (re-do)
3. KAH145(E/X)/ 40 ng ~50 colonies
4. KAH01(E/S)/186, 2 ng + KAH116(E/X)/3975, 20 ng KAH148 1:1 (re-do)
5. KAH02(E/S)/186, 2 ng + " KAH149 1:1 (re-do)
6. KAH03(E/S)/186, 2 ng + " KAH150 1:1 (re-do)
7. KAH04(E/S)/186, 2 ng + " KAH151 1:1 (re-do)
8. KAH06(E/S)/1170, 12 ng + " KAH152 1:1 (re-do)
9. KAH06(E/S)/1173, 12 ng + " KAH153 1:1 (re-do)
10. KAH116(E/X)/ 50 ng ~300 colonies
  1 2 3 4 5 6 7 8 9 10
Insert DNA 1.8 2.8 --- 0.5 0.5 0.5 0.5 0.5 0.5 ---
Vector DNA 2.2 2.2 2.2 0.5 0.5 0.5 0.5 0.5 0.5 0.5
2x lgn buf (Roche) 6.0 6.0 6.0 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
dH2O 1.0 --- 2.8 3.0 3.0 3.0 3.0 3.0 3.0 3.5
  12 μL 12 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL


--> R.T./ ~10 min.; Add to 20 μL Z-DH5α