Team:Hawaii/Notebook/2008-10- 6

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(Difference between revisions)
(New page: {{Team:Hawaii/Header}} = Things we did today = == Wetlab work == ===Construction of secretion device (cont.)=== :<strong> Grace</strong> :*PCR'd nir, nir+rbs, rbs+GFP+tt#2, plac+rbs, slr...)
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::* HindIII/BamHI:
::* HindIII/BamHI:
:::*J33207
:::*J33207
 +
 +
===OriV construction===
 +
:<strong> Margaret</strong>
 +
:*Ligation
 +
::* oriV + pSB1A3 (de-phosphorylated)
 +
 +
:* Transformation into DH5-alpha, with amp100 selection
 +
::* 10/4 ligation
 +
::* 10/6 ligation
 +
::* (+) control pUC18
 +
::* (-) control no plasmid
 +
 +
:* PCR verification
 +
::* colonies 5 and 6 were PCR verified
 +
::* a gel was run
 +
:::* oriV1---> band at 400 bp
 +
:::* oriV2---> no band
 +
:::* oriV3---> no band
 +
:::* oriV4---> no band
 +
:::* oriV5---> band at 700 bp
 +
:::* oriV6---> band at 1000 bp
 +
:::* rep 2---> band at 3000 bp
 +
:::* plac/rbs/rep (colony 8)---> band <3kb (need to re-do this)
 +
 +
 +
===plac/rbs/rep and plac/rbs/aada ===
 +
:<strong> Margaret</strong>
 +
 +
:* plasmid prep of both constructs
 +
:* Restriction digest for 2 hours at 37&deg;C, then a gel was run to verify the digest
 +
::*rep2 with X,P
 +
::*plac/rbs/rep (colony 8) with X,P
 +
::*plac/rbs/rep (colony 8) with E,S
 +
::* plac/rbs/aada with X,P
 +
:*PCR verification of more plac/rbs/rep colonies (14 total) and also colony 8 again to verify that I have what I think I do. (+) was pSB1A3, (-) was water
== Drylab Work ==
== Drylab Work ==

Latest revision as of 08:44, 7 October 2008

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Notebook (t) Meetings (t)

Things we did today

Wetlab work

Construction of secretion device (cont.)

Grace
  • PCR'd nir, nir+rbs, rbs+GFP+tt#2, plac+rbs, slr1+GFPf
  • Verified PCR products on 0.8% agarose gel
  • Overnight RE digest:
  • EcoRI/SpeI:
  • nir
  • nir+rbs
  • plac+rbs
  • XbaI/PstI;
  • slr1+GFPf
  • pilA+GFPf+tt
  • rbs+GFP+tt#2
  • HindIII/BamHI:
  • J33207

OriV construction

Margaret
  • Ligation
  • oriV + pSB1A3 (de-phosphorylated)
  • Transformation into DH5-alpha, with amp100 selection
  • 10/4 ligation
  • 10/6 ligation
  • (+) control pUC18
  • (-) control no plasmid
  • PCR verification
  • colonies 5 and 6 were PCR verified
  • a gel was run
  • oriV1---> band at 400 bp
  • oriV2---> no band
  • oriV3---> no band
  • oriV4---> no band
  • oriV5---> band at 700 bp
  • oriV6---> band at 1000 bp
  • rep 2---> band at 3000 bp
  • plac/rbs/rep (colony 8)---> band <3kb (need to re-do this)


plac/rbs/rep and plac/rbs/aada

Margaret
  • plasmid prep of both constructs
  • Restriction digest for 2 hours at 37°C, then a gel was run to verify the digest
  • rep2 with X,P
  • plac/rbs/rep (colony 8) with X,P
  • plac/rbs/rep (colony 8) with E,S
  • plac/rbs/aada with X,P
  • PCR verification of more plac/rbs/rep colonies (14 total) and also colony 8 again to verify that I have what I think I do. (+) was pSB1A3, (-) was water

Drylab Work

Sequencing analysis

Grace
  • Correct:
  • rbs+GFP+tt #1, 2
  • BBpRL1383a:
  • All four sequenced colonies had a G->C single bp substitution at position 229 of part (in CAP binding site)
  • Colony 18 confirmed (best quality sequence)
  • Colony 1 confirmed (abiguities at 225, 583, 591, 552 of part)
  • Colony 3 (abiguities at 530, 513 of part)
  • Colony 13 (low quality sequence, but BioBrick MCS is present)
  • Incorrect:
  • nir+rbs+GFP #4, 7
  • nir+rbs+slr1+GFPf #8
  • No sequence obtained:
  • plac+rbs
  • nir+rbs+GFP #1
  • nir+rbs+slr1+GFPf #2
  • nir+rbs+pilA #17


Discussion

Quote of the Day

History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson


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