Team:Hawaii/Notebook/2008-10-10

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(New page: {{Team:Hawaii/Header}} = Things we did today = == Wetlab work == ===verification of Transformants=== :<strong> Grace</strong> [[Image:101008colonypcr.jpg|right|thumb|500px|EtBr stained 2%...)
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= Things we did today =
= Things we did today =
== Wetlab work ==
== Wetlab work ==
-
===verification of Transformants===
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===Verification of Transformants===
:<strong> Grace</strong>
:<strong> Grace</strong>
[[Image:101008colonypcr.jpg|right|thumb|500px|EtBr stained 2% agarose gel ran at 95V for 1 hour. Two microliters of PCR product were loaded into each well.]]
[[Image:101008colonypcr.jpg|right|thumb|500px|EtBr stained 2% agarose gel ran at 95V for 1 hour. Two microliters of PCR product were loaded into each well.]]
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:* Colony PCR of transformants
:* Colony PCR of transformants
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::* nrgt #15
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===Overnight RE digest===
 +
:<strong>Grace</strong>
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:* EcoRI/PstI:
 +
::* BBpRL1383a-1
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::* nrsg #6 (PCR)
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::* prpgt #5, 7, 11 (PCR)
===Triparental Conjugation===
===Triparental Conjugation===
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:* Placed plates in SC 37C CO<sub>2</sub> incubator until Monday
:* Placed plates in SC 37C CO<sub>2</sub> incubator until Monday
 +
===Verification of plasmids===
 +
:<strong> MARGARET</strong>
 +
[[Image:plasmid_veri_10_10.jpg|right|thumb|150px|Verification of plasmid preps and digests.]]
 +
 +
:*Why haven't I gotten good efficiency of transformation? I ran a gel of the plasmids i am working with and got the answer. Apparently there was a mix-up and I was using low quality plasmid.
 +
:*The pSB1A3 and pSB3K3 digested and de-phosphorylated (lanes: ) were thrown out. Norman's pSB1A3 and pSB1A7 were digested with E and P today at 4:30pm.
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:* The base vector is digested--> bands appear to be correct size.
 +
 +
===Submitted sequencing===
 +
:<strong> Margaret</strong>
 +
 +
:*the plac/rbs/rep colony 7 construct and oriV colony 1 (from most recent transformations) were sent in for sequencing today.
= Discussion =
= Discussion =

Latest revision as of 03:27, 11 October 2008

Projects Events Resources
Sponsors Experiments Milestones Protocols
Notebook (t) Meetings (t)

Things we did today

Wetlab work

Verification of Transformants

Grace
File:101008colonypcr.jpg
EtBr stained 2% agarose gel ran at 95V for 1 hour. Two microliters of PCR product were loaded into each well.
Construct Colonies
nir+rbs+GFP+tt#1 100
plac+rbs+GFP+tt#1 3
nir+rbs+slr1+GFPf 51
nir+rbs+pilA+GFPf+tt 24
BBpRL1383a lawn
(-) no DNA 0
  • Colony PCR of transformants
  • nrgt #15

Overnight RE digest

Grace
  • EcoRI/PstI:
  • BBpRL1383a-1
  • nrsg #6 (PCR)
  • prpgt #5, 7, 11 (PCR)

Triparental Conjugation

Grace
  • Began triparental conjugation between E. coli containing RP1 and BBpRL1383a -1 or -18 and Synechocystis
  • RP1 OD600=0.6341
  • BBpRL1383-a OD600=61.25
  • BBpRL1383a-18 OD600=0.4884
  • Synechocystis OD700=0.8629, OD730=0.7337
  • Placed plates in SC 37C CO2 incubator until Monday

Verification of plasmids

MARGARET
Verification of plasmid preps and digests.
  • Why haven't I gotten good efficiency of transformation? I ran a gel of the plasmids i am working with and got the answer. Apparently there was a mix-up and I was using low quality plasmid.
  • The pSB1A3 and pSB3K3 digested and de-phosphorylated (lanes: ) were thrown out. Norman's pSB1A3 and pSB1A7 were digested with E and P today at 4:30pm.
  • The base vector is digested--> bands appear to be correct size.

Submitted sequencing

Margaret
  • the plac/rbs/rep colony 7 construct and oriV colony 1 (from most recent transformations) were sent in for sequencing today.

Discussion

Quote of the Day

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


[http://manoa.hawaii.edu/ Sponsor_UHM.gif][http://manoa.hawaii.edu/ovcrge/ Sponsor_OVCRGE.gif][http://www.ctahr.hawaii.edu Sponsor_CTAHR.gif]


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