Team:Hawaii/Notebook/2008-08-14

From 2008.igem.org

(Difference between revisions)
(Wetlab work)
(Restriction Digest)
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:<strong>Grace</strong>
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[[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 loade dinto each well.]]
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:* Checked Krystle's RE digests from last night on a gel
:* Checked Krystle's RE digests from last night on a gel
:* RE digested:
:* RE digested:
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:* Ran RE digests on a gel to purify segments of interest
:* Ran RE digests on a gel to purify segments of interest
::* Also ran RE digested B0015 from Krystle
::* Also ran RE digested B0015 from Krystle
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[[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 loade dinto each well.]]
== Drylab Work ==
== Drylab Work ==

Revision as of 09:14, 15 August 2008

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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.

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.

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
  • RE digested:
  • 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
EtBr stained 2% agarose gel ran at 72V for 1.5 hours. Thirty microliters of the RE digests were loade dinto each well.

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 - KGB


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