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

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

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
1/17/11 E/P digest
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   1/17/11 cloning digest
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


Personal tools