User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/31

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07/31/11

  • ✓ Cultures/ minipreps: KAH228, 229, 230 (cultures from yesterday, 2 each)
  • ✓ Assemblies: pSB31 set, retry with more insert/adpater



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. KAH228 = 2000
3,4. KAH229 = 1756
5,6. KAH230 = 808
Miniprep digests 7/31/11
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
--> Too much background last time. Re-try with dephosphorylated pSB31(E/NcoI)

  1. KAH219/pSB31: KAH219/(E/S)/2422 ✓ + pSB31 BB adapter 1/(S/NcoI)/18 + (4) pSB31/(E/NcoI dp)/5127
  2. KAH220/pSB31: KAH220/(E/S)/1345 ✓ + "
  3. KAH221/pSB31: KAH221/(E/S)/1645 ✓ + "
  4. KAH222/pSB31: KAH222/(E/S)/2614 ✓ + "
  5. KAH223/pSB31: KAH223/(E/S)/1537 ✓ + "
  6. KAH224/pSB31: KAH224/(E/S)/1837 ✓ + "

--> Use 2x as much insert as before --> Dilute stock adapter to 85 ng/μL (use ~10x as much as before)

> Ligations

Ligation Plate results (lig : neg crtl) 08/01/11
1. KAH219(E/S)/2422, 19 ng + pSB31 BB adapter 1(S/NcoI)/18, 0.1 ng + pSB31(E/NcoI dp)/5127, 20 ng KAH219/pSB31 10:1
2. KAH220(E/S)/1345, 11 ng + " KAH220/pSB31 10:1
3. KAH221(E/S)/1645, 13 ng + " KAH221/pSB31 10:1
4. KAH222(E/S)/2614, 20 ng + " KAH222/pSB31 10:1
5. KAH223(E/S)/1537, 12 ng + " KAH223/pSB31 10:1
6. KAH224(E/S)/1837, 14 ng + " KAH224/pSB31 10:1
7. pSB31(E/NcoI)/ 20 ng one colony
  1 2 3 4 5 6 7
Insert DNA 2.9 0.8 1.0 2.0 2.0 1.2 ---
adapter DNA 0.3 0.3 0.3 0.3 0.3 0.3 ---
Vector DNA 0.8 0.8 0.8 0.8 0.8 0.8 0.8
2x lgn buf (Roche) 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
dH2O --- 2.1 1.9 0.9 0.9 1.7 3.2
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL

--> Add 40 μL DH5α Turbo; plate on 100 μg/mL Amp




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