User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/29

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07/29/11

  • ✓ Cultures/ minipreps: KAH219-224/pSB31 (retrial, 2 each)
  • ✓ Assemblies: KAH228 - 231



Minipreps
> Check with E/P digests

Reagent Volume Expected:
pSB31 = 5127
1-3. KAH219/pSB31 = 2422
4-6. KAH220/pSB31 = 1345
7-9. KAH221 = 1645
10-12. KAH222 = 2614
13-15. KAH223 = 1537
16-18. KAH224 = 1837
Miniprep digests
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.


--> None worked. Try again with more insert + adapter.


Assemblies

  1. KAH228: KAH54/(S/P)/430 + (1) KAH226/(X/P)/1564
  2. KAH229: hPCD/(S/P)/186 + "
  3. KAH230: (2) KAH227/(S/P)/577 + polyA2/(X/P)/225


> Digests (Fermentas FD)

Reagent Volume   Cloning digests 7/29/11
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. KAH226 (X/P) 1.099 1.85 55.0
2. KAH227 (S/P) 0.257 1.88 12.9


> Ligations

Ligation Plate results (lig : neg crtl) 07/30/11
1. KAH226(X/P)/1564, 17 ng + KAH54(S/P)/~3630, 20 ng KAH228 >10:1 (Pick 2)
2. KAH54(S/P)/ 20 ng  
3. KAH226(X/P)/1564, 18 ng + hPCD(S/P)/~3386, 20 ng KAH229 >10:1 (Pick 2)
4. hPCD(S/P)/ 20 ng  
5. polyA2(X/P)/225, 1 ng + KAH227(S/P)/~3777, 20 ng KAH230 >10:1 (Pick 2)
6. KAH227(S/P)/ 20 ng  
  1 2 3 4 5 6
Insert DNA 0.5 --- 0.5 --- 0.5 ---
Vector DNA 1.0 1.0 0.3 0.3 1.5 1.5
2x lgn buf (Roche) 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
dH2O 2.5 3.0 3.2 3.7 2.0 2.5
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL

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



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