The printable version is no longer supported and may have rendering errors. Please update your browser bookmarks and please use the default browser print function instead.
Karmella's BioBrick Cloning
|
<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>
|
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)
- ggPro-1, VF2 - correct sequence, scarless junction between hPCD and BL01
- ggPro-1, VR - correct sequence for SP1AB of BL01
- ggPro-2, VF2 - matches ggPro-1 (BLAST)
- ggPro-2, VR - matches ggPro-1 (BLAST)
- ggPro-4, VF2 - matches ggPro-1 (BLAST)
- ggPro-4, VR - matches ggPro-1 (BLAST)
|