User:Karmella Haynes/Notebook/BioBrick cloning/2010/08/07

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08/07/10

  • ✓ Assemblies: KAH160-170 into KAH015 (lentivirus vector)



Assemblies

  1. KAH160/KAH015: (1) KAH160/(E/S)/1147 + (12) KAH015/(E/X)/8402
  2. KAH161/KAH015: (2) KAH161/(E/S)/1147 + "
  3. KAH162/KAH015: (3) KAH162/(E/S)/1147 + "
  4. KAH163/KAH015: (4) KAH165/(E/S)/2131 + "
  5. KAH164/KAH015: (5) KAH164/(E/S)/2134 + "
  6. KAH165/KAH015: (6) KAH165/(E/S)/991 + "
  7. KAH166/KAH015: (7) KAH166/(E/S)/991 + "
  8. KAH167/KAH015: (8) KAH167/(E/S)/991 + "
  9. KAH168/KAH015: (9) KAH168/(E/S)/1955 + "
  10. KAH169/KAH015: (10) KAH169/(E/S)/1958 + "
  11. KAH170/KAH015: (11) KAH170/(E/S)/955 + "


> Digests (Fermentas FD)

Reagent Volume   Hover name
30 μL/lane, 1% agarose
DNA (plasmid) 15.0
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 10.0
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH160 (E/S) 0.071 9.24 3.5
2. KAH161 (E/S) 0.158 4.44 7.9
3. KAH162 (E/S) 0.165 2.99 8.3
4. KAH163 (E/S) 0.204 2.83 10.2
5. KAH164 (E/S) 0.161 3.44 8.0
6. KAH165 (E/S) 0.182 2.75 9.1
7. KAH166 (E/S) 0.138 5.11 6.9
8. KAH167 (E/S) 0.156 2.17 7.8
9. KAH168 (E/S) 0.124 2.22 6.2
10. KAH169 (E/S) 0.086 2.05 4.3
11. KAH170 (E/S) 0.122 3.47 6.1
12. KAH015 (E/X) 0.484 2.11 24.2


> Dephosphorylation (Roche)
--> KAH015(E/X)

Reagent Volume
DNA (clean digest) 17.0 (~400 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~20 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) 08/08/10
1. KAH160(E/S)/1147, 8 ng + KAH015(E/X)/8402, 30 ng KAH160/KAH015 >10:1 (Pick 1)
2. KAH161(E/S)/1147, 8 ng + " KAH161/KAH015 >10:1 (Pick 1)
3. KAH162(E/S)/1147, 8 ng + " KAH162/KAH015 >10:1 (Pick 1)
4. KAH163(E/S)/2131, 15 ng + " KAH163/KAH015 >10:1 (Pick 1)
5. KAH164(E/S)/2134, 15 ng + " KAH164/KAH015 >10:1 (Pick 1)
6. KAH165(E/S)/991, 7 ng + " KAH165/KAH015 >10:1 (Pick 1)
7. KAH166(E/S)/991, 7 ng + " KAH166/KAH015 >10:1 (Pick 1)
8. KAH167(E/S)/991, 7 ng + " KAH167/KAH015 >10:1 (Pick 1)
9. KAH168(E/S)/1955, 14 ng + " KAH168/KAH015 >10:1 (Pick 1)
10. KAH169(E/S)/1958, 14 ng + " KAH169/KAH015 >10:1 (Pick 1)
11. KAH170(E/S)/955, 7 ng + " KAH170/KAH015 >10:1 (Pick 1)
11. KAH015(E/X), 30 ng + " 0 colonies
  1-11 12
Insert DNA 3.5 ---
Vector DNA 1.5 1.5
2x lgn buf (Roche) 6.0 6.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 3.5
  12μL 12μL


--> R.T./~10 min.; Add to 50 μL T-DH5α cells

8/08/10
--> Many colonies on the ligation plates, none on the negative control. Looks great! Pick one colony from each plate; confirm via digest.


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