Team:Hawaii/Notebook/2008-08-14

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== Wetlab work ==
== Wetlab work ==
===Colony PCR===
===Colony PCR===
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[[Image: aada_verification_8_14.jpg|right|thumb|300px|PCR verification of aadA from pRL1383a using wrong conditions.]][[Image:rep_P1_omega_colony_pcr.jpg|right|thumb|300px|PCR verification of rep, P1 lytic regbion, and omega interposon ligation from 8-13]]
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[[Image: aada_verification_8_14.jpg|right|thumb|150px|PCR verification of aadA from pRL1383a using wrong conditions.]][[Image:rep_P1_omega_colony_pcr.jpg|right|thumb|150px|PCR verification of rep, P1 lytic regbion, and omega interposon ligation from 8-13]]
:<strong> Margaret </strong>
:<strong> Margaret </strong>
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:*I ran another PCR verification on rep, P1, and the omega interposon and afterwards a gel.
:*I ran another PCR verification on rep, P1, and the omega interposon and afterwards a gel.
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:<strong>Krystle</strong>
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:* '''Krystle update'''
===Culture===
===Culture===
-
<strong>Margaret</strong>
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:<strong>Margaret</strong>
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:*Plasmid preps from [[Team:Hawaii/Notebook/2008-08-14|yesterday]] did not yield very high quantities. I am setting up the culture for oriV1-4 and aadA(BB) 4 so I can do another plasmid prep tomorrow.
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:*Plasmid preps from [[Team:Hawaii/Notebook/2008-08-14 | yesterday]] did not yield very high quantities. I am setting up the culture for oriV1-4 and aadA(BB) 4 so I can do another plasmid prep tomorrow.
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:<strong>Krystle</strong>
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:* Inoculated for plasmid prep of GFPf and J04430 tomorrow.
===Restriction Digest===
===Restriction Digest===
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:<strong>Margaret</strong>
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<strong>Margaret</strong>
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:*digest of I14032 with SpeI and PstI. I want to use this to ligate to the aadA(BB) construct.
:*digest of I14032 with SpeI and PstI. I want to use this to ligate to the aadA(BB) construct.
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 +
:<strong>Grace</strong>
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:* Checked Krystle's RE digests from last night on a gel
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::* Also ran J33207 PCR product on same gel
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::* Excised J33207 and GFPf bands from gel
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:* RE digested: [[Image:081408REdigests.jpg|right|thumb|200px|EtBr stained 2% agarose gel ran at 72V for 1.5 hours. Thirty microliters of the RE digests were loaded into each well. BB-pRL1383a was digested with EcoRI/PstI. Picture is mislabeled.]]
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::* J33207, J04430, BB-pRL1383a with EcoRI and PstI
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::* pRL1383a, J33207 with HindIII and BamHI (Roche, from SC)
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::* BB-pRL1383a with HindIII and BamHI (from lab -20C)
 +
:* Ran RE digests on a gel to purify segments of interest
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::* Also ran RE digested B0015 from Krystle
 +
 +
===Gel extraction===
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:<strong>Krystle</strong>
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 +
:* Used Epoch Biolabs kit to extract J33207 and GFPf DNA from agarose gel
== Drylab Work ==
== Drylab Work ==
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= Discussion =
= Discussion =
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:* Gel box 1 is the only one that works properly. The unlabeled gel box and box #2 may cause gels to run excessively slow, bands to blur/smear, or other weird things. Amps is really low on these boxes. All covers/power sources are good.
= Quote of the Day =
= Quote of the Day =
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<blockquote>''happy chickens taste better'' - KGB</blockquote>
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<blockquote>''Happy chickens taste better.'' - KSGSK</blockquote>
{{Team:Hawaii/Footer}}
{{Team:Hawaii/Footer}}
__NOTOC__
__NOTOC__

Latest revision as of 19:16, 15 August 2008

Projects Events Resources
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Notebook (t) Meetings (t)

Things we did today

Wetlab work

Colony PCR

PCR verification of aadA from pRL1383a using wrong conditions.
File:Rep P1 omega colony pcr.jpg
PCR verification of rep, P1 lytic regbion, and omega interposon ligation from 8-13
Margaret
  • 5 colonies of rep, P1 lytic region, and aadA(pRL1383a) region, each. pSB1A3 was used as a control.
  • I changed the annealing time to 3min 30 seconds instead of the extension time, so the sites were not amplified properly. I ran the aadA(pRL1383a) construct on a gel and got a few bands, none of which were the right size. One of them should have been right though, so I am assuming this ligation did not work.
  • I ran another PCR verification on rep, P1, and the omega interposon and afterwards a gel.
Krystle
  • Krystle update

Culture

Margaret
  • Plasmid preps from yesterday did not yield very high quantities. I am setting up the culture for oriV1-4 and aadA(BB) 4 so I can do another plasmid prep tomorrow.
Krystle
  • Inoculated for plasmid prep of GFPf and J04430 tomorrow.

Restriction Digest

Margaret
  • digest of I14032 with SpeI and PstI. I want to use this to ligate to the aadA(BB) construct.
Grace
  • Checked Krystle's RE digests from last night on a gel
  • Also ran J33207 PCR product on same gel
  • Excised J33207 and GFPf bands from gel
  • RE digested:
    EtBr stained 2% agarose gel ran at 72V for 1.5 hours. Thirty microliters of the RE digests were loaded into each well. BB-pRL1383a was digested with EcoRI/PstI. Picture is mislabeled.
  • J33207, J04430, BB-pRL1383a with EcoRI and PstI
  • pRL1383a, J33207 with HindIII and BamHI (Roche, from SC)
  • BB-pRL1383a with HindIII and BamHI (from lab -20C)
  • Ran RE digests on a gel to purify segments of interest
  • Also ran RE digested B0015 from Krystle

Gel extraction

Krystle
  • Used Epoch Biolabs kit to extract J33207 and GFPf DNA from agarose gel

Drylab Work

Sequencing

Margaret
  • Verified sequence of oriT, inserted into pSB1A2. The sequences of oriT and the BioBrick sites are correct.

Oligonucleotide Design

Margaret & Grace
  • We are having trouble with TT, RBS and Promoter ligations, so we decided to synthesize these parts.
  • B0016, I14032, B0030, & B0034

Discussion

  • Gel box 1 is the only one that works properly. The unlabeled gel box and box #2 may cause gels to run excessively slow, bands to blur/smear, or other weird things. Amps is really low on these boxes. All covers/power sources are good.

Quote of the Day

Happy chickens taste better. - KSGSK


[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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