User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/17

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)

Reagent	Volume	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)	15 μL/lane; 1% agarose; Ladder
DNA(plasmid)	3.0 μL
10X buffer	1.5
EcoRI	1.0
PstI	1.0
dH2O	9.5
	15 μL --> 37°C/ ~15 min.

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)

1. ggPro-1, VF2 - correct sequence, scarless junction between hPCD and BL01
2. ggPro-1, VR - correct sequence for SP1AB of BL01
3. ggPro-2, VF2 - matches ggPro-1 forward (BLAST)
4. ggPro-2, VR - matches ggPro-1 reverse (BLAST)
5. ggPro-4, VF2 - matches ggPro-1 forward (BLAST)
6. ggPro-4, VR - matches ggPro-1 reverse (BLAST)