User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/28

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

07/28/11

  • ✓ Cultures/ minipreps: KAH226, 227 (2 each)
  • ✓ Assemblies: retry pSB31 ligations



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. KAH226 = 1564
3,4. KAH227 = 577
File:KAH 072811 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
--> 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 ✓ + "


> Dephosphorylation (Roche)

Reagent Volume
DNA (clean digest) 10.7 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O 6.3
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) 07/28/11
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 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 0.8 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 1.3 2.3 2.2 1.7 1.7 2.1 3.2
  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