User:Karmella Haynes/Notebook/BioBrick cloning/2010/04/14

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04/14/10

  • ✓ Minipreps: KAH96, 142, 146, 147/V0201 (12 total); KAH154-159 (12 total)
  • ✓ Assemblies: KAH154-159/MV2
  • ✓ Cultures: KAH142(6), 146(6), 147/V0201(8)
  • ✓ Plasmid transformations: MV2 and V0201



Minipreps
> Check KAH96, 142, 146, 147/V0201 with XbaI digest (add 641 to expected insert size)
> Check KAH154-159 with E/P digest

Reagent Volume Expected:
V0201 band = 4516
Insert band = BB size + 641 bp
1-3. KAH96/V0201 = 3466
4,5. KAH142/V0201 = 5212
6-8. KAH146/V0201 = 7027
9-12. KAH147/V0201 = 8773
---
13,14. KAH154 = 1724
15,16. KAH155 = 1724
17,18. KAH156 = 1724
19,20. KAH157 = 1724
21,22. KAH158 = 2688
23,24. KAH159 = 2691
File:KAH 041410 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.


--> Pick more colonies for KAH142, 146, 147/V0201



Assemblies

  1. KAH154/MV2: (1) KAH154/(E/P)/1724 + (7) MV2/(E/P)/4529
  2. KAH155/MV2: (2) KAH155/(E/P)/1724 + "
  3. KAH156/MV2: (3) KAH156/(E/P)/1724 + "
  4. KAH157/MV2: (4) KAH157/(E/P)/1724 + "
  5. KAH158/MV2: (5) KAH158/(E/P)/2688 + "
  6. KAH159/MV2: (6) KAH159/(E/P)/2691 + "

> Digests (Fermentas FD)
--> Triple digest KAH158, 159 w/ E/P/ScaI
--> Digest for 1 hour to ensure complete digestion

Reagent 1-4, 7 5, 6
DNA (plasmid) up to 25 μL up to 25 μL
10x buffer 3.0 3.0
enzyme 1 1.0 1.0
enzyme 2 1.0 1.0
ScaI --- 1.0
dH2O --- ---
  30 μL 30 μL --> 37°C/ ~1 hr.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH154 (E/P) 0.776 1.86 38.8
2. KAH155 (E/P) 0.869 1.88 43.4
3. KAH156 (E/P) 0.873 1.88 43.6
4. KAH157 (E/P) 0.875 1.90 43.7
5. KAH158 (E/P) 0.753 1.84 37.6
6. KAH159 (E/P) 0.854 1.93 42.7
7. MV2 (E/P) 0.467 2.10 23.4


> Dephosphorylation (Roche)

  1. MV2 (E/P)
Reagent Volume
DNA (clean digest) 21.5 (~500 ng)
10x buffer d.p. 2.5
phosphatase 1.0
dH2O ---
  25 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 20 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH154(E/P)/1724, 15 ng + MV2(E/P)/4529, 20 ng KAH154/MV2 #:1 (Pick #)
2. KAH155(E/P)/1724, 15 ng + " KAH155/MV2 #:1 (Pick #)
3. KAH156(E/P)/1724, 15 ng + " KAH156/MV2 #:1 (Pick #)
4. KAH157(E/P)/1724, 15 ng + " KAH157/MV2 #:1 (Pick #)
5. KAH158(E/P)/2688, 24 ng + " KAH158/MV2 #:1 (Pick #)
5. KAH159(E/P)/2688, 24 ng + " KAH159/MV2 #:1 (Pick #)
2. MV2(E/P)/ 20 ng  
  1 2 3 4 5 6 7
Insert DNA 0.5 0.5 0.5 0.5 1.0 1.0 ---
Vector DNA 1.0 1.0 1.0 1.0 1.0 1.0 1.0
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 2.5 2.5 2.5 2.5 2.0 2.0 3.0
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL


--> R.T./ ~10 min.; Add 28 μL Turbo DH5α (T-DH5α); ice/ 5 min.
--> plate on warm LB, 100 μg/mL Amp; R.T. overnight; 37 °C next day for ~3 hours