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 Karmella's BioBrick Cloning
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01/17/13
- Minipreps: check Golden Gate assembly clones (trial 3)
Minipreps
- Check with E/P digests
- Minipreps from colonies on Plate #2 (Promega home-made ligation buffer trial)
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Volume
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Expected:
1. Plate 2-1 = 2000 (v), 2706 (i)
2. Plate 2-2 = 2000 (v), 2706 (i)
3. Plate 2-3 (red) = 2000 (v), 2706 (i)
4. Plate 2-4 = 2000 (v), 2706 (i)
5. pSB1A3+RFP = 2000 (v), ~1200 (i)
6. hPCD/V0120 = 3200 (v), 186 (i)
7. BL01 = 3200 (v), 2520 (i)
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15 μL/lane; 1% agarose; Ladder
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| DNA(plasmid) |
3.0 μL
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| 10X buffer |
1.5
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| EcoRI |
1.0
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| PstI |
1.0
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| dH2O |
9.5
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15 μL --> 37°C/ ~15 min.
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Conclusions
- Success! (1, 2, & 4) Bands match expected size for vector pSB1A3 (not old vector V0120) and for assembled insert: hPCD (186) + BL01 (2520) = 2706 (visibly larger than BL01 band)
- Transferring inserts from V0120 to pSB1A3 is probably a good idea since several internal BsmBI sites might destroy any template background
- Use BsmBI Type IIS method to assemble all the things!
1/23/13 Sequencing confirmation (DNASU)
- ggPro-1, VF2 - correct sequence, scarless junction between hPCD and BL01
- ggPro-1, VR -
- ggPro-2, VF2 - matches ggPro-1 (BLAST)
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Karmella's BioBrick Cloning
