Difference between revisions of "User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/17"

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
 
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
 
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1/23/13 Sequencing confirmation (DNASU)
 
1/23/13 Sequencing confirmation (DNASU)
 
# ggPro-1, VF2 - correct sequence, scarless junction between hPCD and BL01
 
# ggPro-1, VF2 - correct sequence, scarless junction between hPCD and BL01
# ggPro-1, VR -  
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# ggPro-1, VR - correct sequence for SP1AB of BL01
# ggPro-2, VF2 - matches ggPro-1 (BLAST)
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# ggPro-2, VF2 - matches ggPro-1 forward (BLAST)
 +
# ggPro-2, VR - matches ggPro-1 reverse (BLAST)
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# ggPro-4, VF2 - matches ggPro-1 forward (BLAST)
 +
#ggPro-4, VR - matches ggPro-1 reverse (BLAST)
  
  

Latest revision as of 21:22, 26 September 2017

Owwnotebook icon.pngKarmella's BioBrick Cloning Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

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)