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
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Volume
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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.
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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
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DNA(BL01x2, BL05x1, BL09x2, CMV/MV9x1) |
3.0 μL
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10X buffer |
1.5 μL
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XbaI |
1.0 μL
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NruI |
1.0 μL
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dH2O |
8.5 μL
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Total |
15 μL --> 37°C/ 30 min.
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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
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Volume
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Expected: 1. BL01, BL05 = 2500bp, BL09=1400bp
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DNA |
25.0 μL
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10X buffer |
3.0 μL
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XbaI |
1.0 μL
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SpeI |
1.0 μL
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dH2O |
0 μL
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Total |
30 μL --> 37°C/ 35 min.
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0.8% agarose; cut the brightest band furthest down at ~ 2500bp; Ladder
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- 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 elution buffer to the column.
- Spun at max rpm for 30 seconds.
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