Team:Hawaii/Notebook/2008-10-17

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(Difference between revisions)
(New page: {{Team:Hawaii/Header}} = Things we did today = == Wetlab work == ===Restreaked BBpRL1383a construct=== :<strong> Grace</strong> :* Looked at J33207+pRL1383a (BBpRL1383a) plates. 50 μl...)
(→Construction of GFP secretion devices)
 
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:* Looked at J33207+pRL1383a (BBpRL1383a) plates. 50 &mu;l plate had blue colonies. Restreaked on LB+sp<sub>2.5</sub>+sm<sub>2.5</sub> + X-gal
:* Looked at J33207+pRL1383a (BBpRL1383a) plates. 50 &mu;l plate had blue colonies. Restreaked on LB+sp<sub>2.5</sub>+sm<sub>2.5</sub> + X-gal
 +
 +
===Inoculated for plasmid prep===
 +
:<strong>Grace</strong>
 +
 +
:* Inoculated 200 ml TB+sp<sub>2.5</sub>+sm<sub>2.5</sub> with blue colony. Incubated over the weekend at 37C with shaking at 250 rpm.
 +
 +
===PCR verification of constructs===
 +
:<strong>Margaret</strong>
 +
 +
:*20 colonies from each of [[Team:Hawaii/Notebook/2008-10-16|yesterday's]] transformation were verified by PCR today using the VF2 and VR primers.
 +
 +
===Construction of GFP secretion devices===
 +
:<strong>Krystle</strong>
 +
:* Checked for transformants:
 +
::*nir+rbs+slr+gfpf
 +
:::competent cells- none
 +
:::commercial competent cells- 4
 +
::*lac+rbs+slr+gfpf
 +
:::competent cells- 1
 +
:::commercial competent cells- 3
 +
:* Colony PCR
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::*nir+rbs+slr+gfpf colony 2 and colony 3 appear to be correct size
 +
:::*lac+rbs+slr+gfpf commercial cells colonies 1, 3, and competent cell colony appear to be correct size
 +
 +
== Drylab work ==
 +
===Sequencing===
 +
:<strong> Margaret</strong>
 +
 +
:*Results for plac/rbs(30)/rep(7) came in. The promoter and rbs were not present. The only discrepancies in the sequence were due to bad sequence and could be explained by overlapping sequences.
 +
:*This construct is already ligated to oriV and the oriT/aada so next step will be to isolate that plasmid, digest with E,S and ligate the promoter and rbs in front. rbs (B0034) will be used because we have primers for this rbs and it can be easily verified.

Latest revision as of 00:45, 30 October 2008

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Things we did today

Wetlab work

Restreaked BBpRL1383a construct

Grace
  • Looked at J33207+pRL1383a (BBpRL1383a) plates. 50 μl plate had blue colonies. Restreaked on LB+sp2.5+sm2.5 + X-gal

Inoculated for plasmid prep

Grace
  • Inoculated 200 ml TB+sp2.5+sm2.5 with blue colony. Incubated over the weekend at 37C with shaking at 250 rpm.

PCR verification of constructs

Margaret
  • 20 colonies from each of yesterday's transformation were verified by PCR today using the VF2 and VR primers.

Construction of GFP secretion devices

Krystle
  • Checked for transformants:
  • nir+rbs+slr+gfpf
competent cells- none
commercial competent cells- 4
  • lac+rbs+slr+gfpf
competent cells- 1
commercial competent cells- 3
  • Colony PCR
  • nir+rbs+slr+gfpf colony 2 and colony 3 appear to be correct size
  • lac+rbs+slr+gfpf commercial cells colonies 1, 3, and competent cell colony appear to be correct size

Drylab work

Sequencing

Margaret
  • Results for plac/rbs(30)/rep(7) came in. The promoter and rbs were not present. The only discrepancies in the sequence were due to bad sequence and could be explained by overlapping sequences.
  • This construct is already ligated to oriV and the oriT/aada so next step will be to isolate that plasmid, digest with E,S and ligate the promoter and rbs in front. rbs (B0034) will be used because we have primers for this rbs and it can be easily verified.


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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