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

From OpenWetWare

< User:Karmella Haynes | Notebook | BioBrick cloning | 2013 | 01(Difference between revisions)
Jump to: navigation, search
(→01/17/13)
Current revision (16:06, 3 March 2013) (view source)
(→01/17/13)
 
(4 intermediate revisions not shown.)
Line 41: Line 41:
* Transferring inserts from V0120 to pSB1A3 is probably a good idea since several internal BsmBI sites might destroy any template background
* 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!
* 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 forward (BLAST)
 +
# ggPro-2, VR - matches ggPro-1 reverse (BLAST)
 +
# ggPro-4, VF2 - matches ggPro-1 forward (BLAST)
 +
#ggPro-4, VR - matches ggPro-1 reverse (BLAST)

Current revision

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

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)
 E/P digests
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)



Retrieved from "http://www.openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/01/17"
Personal tools