Haynes Lab:Notebook/Engineering PC-TFs/2014/07/09

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Summary

Liquid Culture Minipreps
Eluted with 70μL dH2O.


Plasmid OD260 OD260/280 ng/μL
1. BL01 C1 0.289 1.89 288.704
1. BL01 C2 0.463 1.918 462.857
1. BL01 C3 0.495 1.915 495.398


Plasmid OD260 OD260/280 ng/μL
1. BL05 C1 0.047 2.124 46.727
1. BL05 C2 0.301 1.912 301.14
1. BL05 C3 0.075 1.97 74.969


Plasmid OD260 OD260/280 ng/μL
1. BL09 C1 0.059 1.986 59.185
1. BL09 C2 0.236 1.878 235.782
1. BL09 C3 0.185 1.958 184.993



Restriction Digest

Reagent Volume Expected:
BL01(W1:2-4), BL05(W1:5, W2:2-3)
Forward insertion: 710, 7010.
Reverse insertion: 3230, 4490.
No insertion: 710, 4490.
BL09 (W2:4-6)
Forward insertion: 710, 5921
Reverse insertion: 2141, 4490
No insertion: 710, 4490.
*Final well is empty CMV/MV9.
Today's gel
15 μL/lane; 1% agarose;
Observed lengths: W2: 7100, 700; W3: 7100, 5200, 2500; W4: *5200, 2500, 710; W5: 5000, 2500; W6: 5000, 2500, 710.
*Uncut plasmid.
Ladder
Today's gel
15 μL/lane; 1% agarose;
Observed lengths: W2: *7100, 700; W3: *6100, 2500, 710; W4: *7200, 2500, 710; W5: 5000, 4000; W6: *5500, 2500, 750.
*Uncut plasmid.
Ladder
DNA(BL01x3, BL05x3, BL09x3, CMV/MV9) 5.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
NruI 1.0 μL
dH2O 6.5 μL
Total 15 μL --> 37°C/ 30 min.


Results
Top W2: BL01 possible success.
Bottom W2: BL05 possible success.
Bottom W4: BL09 possible success.


Sequencing Results Order 9218
BL01rA

  • Used 1μL BL01 miniprep 1, 1μL DD123 B primer, 8μLdH2O.
  • Results: Matches were found after the primer and then just before CMV suggesting no insert.


BL01rB

  • Used 1μL BL01 miniprep 2, 1μL DD123 B primer, 8μLdH2O.
  • Results: Matches were found after the primer and then just before CMV suggesting no insert.


BL09rA

  • Used 1μL BL09 miniprep 1, 1μL DD123 B primer, 8μLdH2O.
  • Results: Matches were found after the primer and then just before CMV suggesting no insert.


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