User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/25

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07/25/11

  • ✓ Retransformations: 183-DF4, -DF5, -DF6/MV8; 183-177/MV8; STOP
  • ✓ Assemblies A: DPRE-208/V0200; KAH225, KAH226
  • ✓ Assemblies B: miR-luc sensor "198/MV8" set



Assemblies

  1. DPRE-208/V0200: (1) DPRE/(N)/1740 + (2) KAH208/V0200/(N dp)/6326
  2. KAH225: mCh/(S/P)/705 ✓ + (3) p65/(X/P)/783


> Digests (Fermentas FD)

Reagent Volume  
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. DPRE (N) 1.518 1.83 75.9
2. KAH208/V0200 (N) 0.385 1.59 19.2
3. p65 (X/P) 0.359 1.56 18.0


> Dephosphorylation (Roche)

Reagent Volume
DNA (clean digest) up to 17 μL (max, 326.4 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 16 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. DPRE(N)/1740, 11 ng + KAH208/V0200(N dp)/6326, 20 ng DPRE-208/V0200 >10:1 (Pick 4)
2. KAH208/V0200(N dp)/ 20 ng  
3. p65(X/P)/783, 8 ng + mCh(S/P)/~3950, 20 ng KAH225 >10:1 (Pick 2)
4. mCh(S/P)/ 20 ng  
  1 2 3 4
Insert DNA 0.2 --- 0.4 ---
Vector DNA 1.3 1.3 0.4 0.4
2x lgn buf (Roche) 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0
dH2O 2.5 2.7 3.2 3.6
  10 μL 10 μL 10 μL 10 μL



Assemblies B
--> Last time (6/06/11), may have used too much insert. Use calculated amount this time
--> See 7/20/11 for 198/MV8 digest and concentration

  1. KAH186-196/MV8: KAH186/(E/S)/2025 ✓ + KAH198/MV8(E/X)/7978 ✓
  2. KAH184-196/MV8: KAH187/(E/S)/2269 ✓ + "
  3. KAH187-196/MV8: KAH187/(E/S)/2269 ✓ + "
  4. KAH188-196/MV8: KAH188/(E/S)/2124 ✓ + "
  5. KAH189-196/MV8: KAH189/(E/S)/2296 ✓ + "
  6. KAH190-196/MV8: KAH190/(E/S)/2368 ✓ + "
  7. KAH191-196/MV8: KAH191/(E/S)/2223 ✓ + "
  8. KAH183-196/MV8: KAH191/(E/S)/2223 ✓ + "
  9. KAH192-196/MV8: KAH192/(E/S)/2461 ✓ + "


> Ligations

Ligation Plate results (lig : neg crtl) 07/14/11
1-9. KAH###(E/S)/~2200, 11 ng + KAH198/MV8(E/X)/8050, 20 ng Notes: ~5:1 for all plates
10. KAH198/MV8(E/X)/ 20 ng  
  1 2 3 4 5 6 7 8 9 10
Insert DNA 1.0 1.0 1.5 1.0 1.0 0.5 1.0 0.5 0.8 ---
Vector DNA 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4
2x lgn buf (Roche) 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
dH2O 2.6 2.6 2.1 2.6 2.6 3.1 2.6 3.1 2.8 3.6
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL

--> Add to 30 μL DH5α Turbo; plate on 100 μg/mL amp


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