User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/27

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07/27/11

  • ✓ Assemblies: KAH226, 227; 219-224 into pSB31 w/ adapter



Assemblies
> Assemble KAH226, 227 as usual
> Blunt cloning for pSB31 didn't work well. Use EcoRI/NcoI-cut vector, add SpeI-EcoRI-NcoI adapter to E/S insert. Two EcoRI sites will allow quick screening of minipreps.

  1. KAH226: (1) KAH225/(E/S)/1494 + KAH011/(E/X)/64 ✓
  2. KAH227: (2) Gluc/(E/S)/558 + (3) STOP/(E/X)/13
  3. KAH219/pSB31: KAH219/(E/S)/2422 ✓ + pSB31 BB adapter 1/(S/NcoI)/18 + (4) pSB31/(E/NcoI)/5127
  4. KAH220/pSB31: KAH220/(E/S)/1345 ✓ + "
  5. KAH221/pSB31: KAH221/(E/S)/1645 ✓ + "
  6. KAH222/pSB31: KAH222/(E/S)/2614 ✓ + "
  7. KAH223/pSB31: KAH223/(E/S)/1537 ✓ + "
  8. KAH224/pSB31: KAH224/(E/S)/1837 ✓ + "

> Digests (Fermentas FD)
--> Specific notes

Reagent Volume   File:KAH 072711 gel1.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. KAH225 (E/S) 0.867 1.99 43.4
2. Gluc (E/S) 0.280 2.45 14.0
3. STOP (E/X) 0.214 2.25 10.7
4. pSB31 (E/NcoI) 0.931 1.97 46.6

--> pSB31 BB adapter 1 (IDT) = 850 ng/μL (Dilute to 8.5 ng/μL before use)


> Ligations

Ligation Plate results (lig : neg crtl) 07/28/11
1. KAH225(E/S)/1494, 18 ng + KAH011(E/X)/~3264, 20 ng KAH226 >10:1 (Pick 2)
2. KAH011(E/X)/ 20 ng  
3. Gluc(E/S)/558, 7 ng + STOP(E/X)/~3213, 20 ng KAH226 >10:1 (Pick 2)
4. STOP(E/X)/ 20 ng  
5. KAH219(E/S)/2422, 19 ng + pSB31 BB adapter 1(S/NcoI)/18, 0.1 ng + pSB31(E/NcoI)/5127, 20 ng KAH219/pSB31 1:1
6. KAH220(E/S)/1345, 11 ng + " KAH220/pSB31 1:1
7. KAH221(E/S)/1645, 13 ng + " KAH221/pSB31 1:1
8. KAH222(E/S)/2614, 20 ng + " KAH222/pSB31 1:1
9. KAH223(E/S)/1537, 12 ng + " KAH223/pSB31 1:1
10. KAH224(E/S)/1837, 14 ng + " KAH224/pSB31 1:1
11. pSB31(E/NcoI)/ 20 ng  
  1 2 3 4 5 6 7 8 9 10 11
Insert DNA 0.4 --- 0.5 --- 1.4 0.4 0.5 1.0 1.0 0.6 ---
adapter DNA --- --- --- --- 0.5 0.5 0.5 0.5 0.5 0.5 ---
Vector DNA 0.5 0.5 2.0 2.0 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 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 1.0
dH2O 3.1 3.5 1.5 2.0 1.7 2.7 2.6 2.1 2.1 2.5 3.6
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL

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

--> pSB31 ligations failed. Too much background on neg ctrl. Dephosphorylate pSB31(E/NcoI) and try again.