User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/12

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07/12/11

  • ✓ Assemblies: KAH213, 214, 215; KAH216, 217, 218; KAH207, 208, 209/V0200 (re-do with d.p. treated V200)


Assemblies

  1. KAH213: (4) KAH54/(S/P)/430 + (1) KAH210/(X/P)/2398
  2. KAH214: " + (2) KAH211/(X/P)/1321
  3. KAH215: " + (3) KAH212/(X/P)/1621
  4. KAH216: hPCD/(S/P)/186 ✓ + KAH210/(X/P)/2398
  5. KAH217: " + KAH211/(X/P)/1321
  6. KAH218: " + KAH212/(X/P)/1621
  7. KAH207/V0200: KAH207/(N/S)/1836 ✓ + V0200/(N/S dp)/4442
  8. KAH208/V0200: KAH208/(N/S)/1884 ✓ + "
  9. KAH209/V0200: KAH209/(N/S)/1932 ✓ + "


> Digests (Fermentas FD)

Reagent Volume   Cloning digests 7/12/11
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. KAH210 (X/P) 0.142 1.86 7.1
2. KAH211 (X/P) 0.328 2.05 16.4
3. KAH212 (X/P) 0.323 1.78 16.1
4. KAH54 (s/P) 0.492 2.06 24.6


> Dephosphorylation (Roche)
--> V0200 (N/S)

Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) 07/13/11
1. KAH210(X/P)/2398, 33 ng + KAH54(S/P)/~3630, 25 ng KAH213 >10:1 (Pick 2)
2. KAH211(X/P)/1321, 18 ng + " KAH214 >10:1 (Pick 2)
3. KAH212(X/P)/1621, 22 ng + " KAH215 >10:1 (Pick 2)
4. KAH54(S/P)/ 25 ng  
5. KAH210(X/P)/2398, 35 ng + * hPCD(S/P)/~3386, 25 ng KAH216 #:1 (Pick #)
6. KAH211(X/P)/1321, 19 ng + " KAH217 >10:1 (Pick 2)
7. KAH212(X/P)/1621, 24 ng + " KAH218 >10:1 (Pick 2)
8. hPCD(S/P)/ 25 ng  
9. KAH207(N/S)/1836, 21 ng + V0200(N/S dp)/4442, 25 ng KAH207/V0200 >10:1 (Pick 2)
10. KAH208(N/S)/1884, 21 ng + " KAH208/V0200 >10:1 (Pick 2)
11. KAH209(N/S)/1932, 22 ng + " KAH209/V0200 >10:1 (Pick 2)
12. V0200(N/S dp)/ 25 ng  

--> * Dilute hPCD to 25 ng/μL before use

  1 2 3 4 5 6 7 8 9 10 11 12
Insert DNA 5.0 1.5 1.5 --- 5.0 1.5 1.5 --- 1.5 1.0 1.0 ---
Vector DNA 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
2x lgn buf (Roche) 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 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 --- 3.5 3.5 5.0 --- 3.5 3.5 5.0 1.5 2.0 2.0 3.0
  14 μL 14 μL 14 μL 14 μL 14 μL 14 μL 14 μL 14 μL 10 μL 10 μL 10 μL 10 μL

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


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