05/29/15
- David Tze - minpreps & digests
- Ben - sequencing order
- Cas-tone - sequencing order
David Tze - minpreps & digests
- Minipreps
- Sigma GenElute kit, elute w/ 75 μL elution sln.
- Digests
- Check with E/S digests; cannot use PstI because PLrigid & PLrigid4 contain PstI sites
Reagent
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Volume
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Expected: 1. DT001 = 2668, 252 Empty PLflex = 2668, 95 2. DT002 = 2656, 432 Empty PLflex4 = 2656, 274 3. DT003 = 2668, 252 Empty PLrigid = 2668, 95 4. DT004 = 2656, 432 Empty PLrigid4 = 2656, 275
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15 μL/lane; 1% agarose; Ladder
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DNA(plasmid) |
2.0 μL
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10X buffer |
1.5
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EcoRI |
1.0
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SpeI |
1.0
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dH2O |
9.5
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15 μL --> 37°C/ ~15 min.
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CONCLUSIONS:
- DT001 - success.
- DT002 - success.
- DT003 - success.
- DT004 - success.
- Next steps:
- Sequence verify all clones (extra precaution; linker part is small)
Sequencing order
- 1-4 - Ben
- 5-16 - Cas-tone
- DBN007-1 - VF2 - good
- DBN007-1 - VR - good, pSB1A3 terminator appears to be deleted
- DBN007-2 - VF2 - good
- DBN007-2 - VR - good, pSB1A3 terminator appears to be deleted
- H3140-1 - CMVf
- H3140-1 - DD123
- H3140-2 - CMVf
- H3140-2 - DD123
- H3160-1 - CMVf
- H3160-1 - DD123
- H3160-2 - CMVf
- H3160-2 - DD123
- H3180-1 - CMVf
- H3180-1 - DD123
- H3180-2 - CMVf
- H3180-2 - DD123
- H31116-2 - CMVf
- H31116-2 - DD123
- H31136-1 - CMVf
- H31136-1 - DD123
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