User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/17

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01/17/11

  • ✓ Minipreps: KAH173(2), 174(2)
  • ✓ Assemblies: KAH175-178



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. KAH173 = 2960
3,4. KAH174 = 1990
File:KAH 011711 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. KAH175: (1) KAH174/(S/P)/~5190 + polyA2/(X/P)/225 ✓
  2. KAH176: (2) KAH173/(S/P)/~6160 + polyA2/(X/P)/225 ✓
  3. KAH177: " + (3) KAH171/(X/P)/418
  4. KAH178: " + (4) KAH172/(X/P)/429


> Digests (Fermentas FD)

Reagent Volume   File:KAH 011711 gel2.tif
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. KAH174 (S/P) 0.270 1.91 13.52
2. KAH173 (S/P) 0.866 1.82 43.3
3. KAH171 (X/P) 0.251 1.95 12.6
4. KAH172 (X/P) 0.286 1.87 14.3


> Ligations

Ligation Plate results (lig : neg crtl) 01/18/11
1. polyA2(X/P)/225, 2.2 ng + KAH174(S/P)/~5190, 25 ng KAH175 >10:1 (Pick 2)
2. KAH174(S/P)/ 25 ng  
3. polyA2(X/P)/225, 1.8 ng + KAH173(S/P)/~6160, 25 ng KAH175 >10:1 (Pick 2)
4. KAH171(X/P)/418, 3.4 ng + " KAH175 >10:1 (Pick 2)
5. KAH172(X/P)/429, 3.5 ng + " KAH175 >10:1 (Pick 2)
6. KAH173(S/P)/ 25 ng  
  1 2 3 4 5 6
Insert DNA 1.0 --- 1.0 1.0 1.0 ---
Vector DNA 2.0 2.0 0.5 0.5 0.5 0.5
2x lgn buf (Roche) 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
dH2O 1.0 2.0 2.5 2.5 2.5 3.5
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL

--> ~10 min./ rt; add to 30 μL DH5α turbo