Haynes Lab:Notebook/Engineering PC-TFs/2014/07/28

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Summary

  • Ran another gel verification on prior ligation results.
  • Gel extraction/purification for BL01, BL05, BL09.
  • Passaged cells.
  • Digested/dephos'd CMV/MV9.
  • Resubmitted samples for sequencing to biodesign.

Ligation Digest and Gel Verification (2)

Reagent Volume Expected:
BL01(Well:2&3), CMV/MV9 (Well: 4) BL05(Well:5), BL09 (Well: 6&7)
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.
Today's gel
15 μL/lane; 1% agarose;
Observed lengths: W2: 710, 4490; W3: 710, 4490; W4: 710, 4490; W5: 710, 4490; W6: 710, 4490.
W7: 7200, 5000, 2500
Ladder
DNA(BL01x2, BL05x1, BL09x2, CMV/MV9x1) 3.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
NruI 1.0 μL
dH2O 8.5 μL
Total 15 μL --> 37°C/ 30 min.


Sequencing Results Order 9218 (update when receive new sequencing data)



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.


Gel Extraction/Purification



Reagent Volume Expected:
1. BL01, BL05 = 2500bp, BL09=1400bp
DNA 25.0 μL
10X buffer 3.0 μL
XbaI 1.0 μL
SpeI 1.0 μL
dH2O 0 μL
Total 30 μL --> 37°C/ 35 min.


Hover name
0.8% agarose; cut the brightest band furthest down at ~ 2500bp&1400bp; Ladder
  • Used Zymo gel purification kit.
  • Assumed 200mg gel, added 600μL ADB to the 1.5mL tube.
  • Put on heat block at 55°C for 5 minutes, vortexed and then placed back on the heat block for an additional 5 minutes, vortexed again.
  • Put ~750μL gel/ADB into spin column, centrifuged at max rpm for 30 seconds.
  • Pipetted 200μL wash buffer to the tube, centrifuged at max rpm for 30 seconds. Repeated once.
  • Transferred column to new labeled 1.5mL tube.
  • Pipetted 15μL dH2O to the column.
  • Spun at max rpm for 30 seconds.


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