User:Karmella Haynes/Notebook/BioBrick cloning/2010/04/12

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04/12/10

  • ✓ Minipreps: KAH146-153
  • ✓ Assemblies: KAH96, 142, 146, 147/ V0201; KAH154-159



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. KAH146 = 6386
3,4. KAH147 = 8132
5,6. KAH148 = 967
7,8. KAH149 = 967
9,10. KAH150 = 967
11,12. KAH151 = 967
13,14. KAH152 = 1931
15,16. KAH153 = 1934
17. KAH116 (vector ctrl) = 775
E/P digests 4/12/10
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

  1. KAH96/V0201: (1) KAH96/(N/S)/2825 + V0201/(N/S dp)/5157 ✓
  2. KAH142/V0201: (2) KAH142/(N/S)/4571 + "
  3. KAH146/V0201: (3) KAH146/(N/S)/6386 + "
  4. KAH147/V0201: (4) KAH147/(N/S)/8132 + "
  5. KAH154: (5) KAH148/(X/P)/967 + KAH3/(S/P)/~3951 ✓
  6. KAH155: (6) KAH149/(X/P)/967 + "
  7. KAH156: (7) KAH150/(X/P)/967 + "
  8. KAH157: (8) KAH151/(X/P)/967 + "
  9. KAH158: (9) KAH152/(X/P)/1931 + "
  10. KAH159: (10) KAH153/(X/P)/1934 + "


> Digests (Fermentas FD)
--> Triple digest KAH96 w/ N/S/ApaLI
--> Do all digests for at least 1 hour to ensure complete digestion

Reagent Volume   Cloning digests 4/12/10
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/ ~1 hr.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH96 (N/S) 1.062 1.85 53.1
2. KAH142 (N/S) 0.874 1.83 43.7
3. KAH146 (N/S) 0.435 2.14 21.7
4. KAH147 (N/S) 0.313 1.82 15.7
5. KAH148 (X/P) 0.407 1.84 20.3
6. KAH149 (X/P) 0.336 2.11 16.8
7. KAH150 (X/P) 0.406 2.13 20.3
8. KAH151 (X/P) 0.326 2.04 16.3
9. KAH152 (X/P) 0.777 1.90 38.9
10. KAH153 (X/P) 0.557 1.96 27.9

--> Already cut:

  1. V0201 (N/S dp) = 25 ng/μL
  2. KAH3 (S/P) = 27.9 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) 04/13/10
1. KAH96(N/S)/2825, 33 ng + V0201(N/S)/5157, 30 ng KAH96/V0201 5:1 (Pick 3)
2. KAH142(N/S)/4571, 53 ng + " KAH142/V0201 10:1 (Pick 2)
3. KAH146(N/S)/6386, 74 ng + " KAH146/V0201 5:1 (Pick 3)
4. KAH147(N/S)/8132, 95 ng + " KAH147/V0201 2:1 (Pick 4)
5. V0201(N/S)/ 30 ng ~12 colonies
6. KAH148(X/P)/967, 10 ng + KAH3(S/P)/3951, 20 ng KAH154 >10:1 (Pick 2)
7. KAH149(X/P)/967, 10 ng + " KAH155 >10:1 (Pick 2)
8. KAH150(X/P)/967, 10 ng + " KAH156 >10:1 (Pick 2)
9. KAH151(N/S)/967, 10 ng + " KAH157 >10:1 (Pick 2)
10. KAH152(N/S)/1931, 20 ng + " KAH158 >10:1 (Pick 2)
11. KAH153(N/S)/1934, 20 ng + " KAH159 >10:1 (Pick 2)
12. KAH3(S/P)/ 20 ng ~20 colonies


  1 2 3 4 5 6 7 8 9 10 11 12
Insert DNA 0.8 1.8 3.8 3.8 --- 1.3 1.3 1.3 1.3 1.3 1.3 ---
Vector DNA 1.2 1.2 1.2 1.2 1.2 0.7 0.7 0.7 0.7 0.7 0.7 0.7
2x lgn buf (Roche) 6.0 6.0 6.0 6.0 6.0 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 1.0
dH2O 3.0 2.0 --- --- 3.8 2.0 2.0 2.0 2.0 2.0 2.0 3.3
  12 μL 12 μL 12 μL 12 μL 12 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL

--> Ligations 1-5: R.T./~1 hr.; Add to 25 μL Z-DH5α
--> Ligations 6-12: R.T./~10 min.; Add to 25 μL Z-DH5α



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