User:Karmella Haynes/Notebook/BioBrick cloning/2011/08/12

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08/12/11

  • ✓ Cultures/ minipreps: (cultures from last night); Pc-TF constructs in pSB31 (3 each, 24 total)
  • ✓ Assemblies: KAH234, 235/ V0200



Minipreps
> Check with E digests
> Also check with NcoI digests for orientation (Expected sizes are for the forward orientation); p65 (KAH232, 231) has 2 NcoI sites

Reagent Volume Expected:
Gel 1:
Vector = 5127
1-3. KAH222 = 2614
4-6. KAH223 = 1537
7-9. KAH224 = 1837
10-12. KAH232 = 1780
13-15. KAH219 = 2422
16-18. KAH220 = 1345
19-21. KAH221 = 1645
22-24. KAH231 = 1588

Gel 2:
Vector
fwd = ~5000
rev = >5000
*Last NcoI frag + 141
1-3. KAH222 = 2108*, 636
4-6. KAH223 = 1031*, 636
7-9. KAH224 = 1331*, 636
10-12. KAH232 = 513, 99, 662*, 636
13-15. KAH219 = 2108*, 450
16-18. KAH220 = 1031*, 450
19-21. KAH221 = 1331*, 450
22-24. KAH231 = 513, 99, 662*, 450

Miniprep digests 8/12/11
15 μL/lane; 1% agarose

Miniprep digests 2, 8/12/11
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. KAH234/V0200: (1) KAH234/(N/S)/1041 + V0200/(N/S dp)/4442 ✓
  2. KAH235/V0200: (2) KAH235/(N/S)/2787 + "

> Digests (Fermentas FD)

Reagent Volume   Cloning digests, 8/12/11
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. KAH234 (N/S) 0.205 1.66 10.2
2. KAH235 (N/S) 0.242 2.41 12.1


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH234(N/S)/1041, 9 ng + V0200(N/S dp)/4442, 20 ng KAH234/V0200 >10:1 (Pick 2)
2. KAH235(N/S)/2787, 25 ng + " KAH235/V0200 >10:1 (Pick 4)
3. V0200(N/S dp)/ 20 ng  
  1 2 3
Insert DNA 1.0 2.0 ---
Vector DNA 0.8 0.8 0.8
2x lgn buf (Roche) 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0
dH2O 2.2 1.2 3.2
  10 μL 10 μL 10 μL



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