User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/09

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07/09/11

  • ✓ Miniprep digests: check 183-177/MV8 clones #2, 4 (sequence verified)
  • ✓ Assemblies: KAH207, 208, 209/V0200; KAH210, 211, 212



Minipreps
> Check with ApaLI digest

Reagent Volume Expected:
MV8 bands = 1243, 1135, 288
1. KAH183-177/MV8 #2 = 4218*, 2772**, 711
2. KAH183-177/MV8 #4 = 4218*, 2772**, 711
*insert + 234 (vector) + 1601 (177 frag)
**last 177 frag + 1655
Miniprep digests 7/09/11
15 μL/lane; 1% agarose
DNA(plasmid) 5.0 μL
10X buffer 1.5
ApaLI 1.0
dH2O 7.5
  15 μL --> 37°C/ ~15 min.



Assemblies

  1. KAH207/V0200: (1) KAH207/(N/S)/1836 + (4) V0200/(N/S)/4442
  2. KAH208/V0200: (2) KAH208/(N/S)/1884 + "
  3. KAH209/V0200: (3) KAH209/(N/S)/1932 + "
  4. KAH210: (5) KAH204/(E/S)/2328 + NLS myc stop/(E/X)/64 ✓
  5. KAH211: (6) KAH205/(E/S)/1251 + "
  6. KAH212: (7) KAH206/(E/S)/1551 + "

> 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. KAH207 (N/S) 0.333 2.27 16.7
2. KAH208 (N/S) 0.484 2.04 24.2
3. KAH209 (N/S) 0.467 2.11 23.4
4. V0200 (N/S) 2.266 1.83 113.3 (diluted to 50 ng/μL w/ EB)
5. KAH204 (N/S) 0.205 2.01 10.3
6. KAH205 (N/S) 0.401 1.78 20.0
7. KAH206 (N/S) 0.423 1.72 21.1


> Ligations

Ligation Plate results (lig : neg crtl) 07/10/11
1. KAH207(N/S)/1836, 21 ng + V0200(N/S)/4442, 25 ng KAH207/V0200 1:1 (re-do*)
2. KAH208(N/S)/1884, 21 ng + " KAH208/V0200 1:1 (re-do*)
3. KAH209(N/S)/1932, 22 ng + " KAH209/V0200 1:1 (re-do*)
4. V0200(N/S)/ 25 ng  
5. KAH204(E/S)/2328, 35 ng + NLS myc stop(E/X)/~3264, 25 ng KAH210 >10:1 (Pick 2)
6. KAH205(E/S)/1251, 19 ng + " KAH211 >10:1 (Pick 2)
7. KAH206(E/S)/1551, 24 ng + " KAH212 >10:1 (Pick 2)
  1 2 3 4 5 6 7 8
Insert DNA 1.5 1.0 1.0 --- 3.4 1.0 1.5 ---
Vector DNA 0.5 0.5 0.5 0.5 0.6 0.6 0.6 0.6
2x lgn buf (Roche) 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
dH2O 2.0 2.5 2.5 3.5 --- 2.4 1.9 3.4
  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

--> * Re-do V0200 ligations using dephosphorylated vector


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