Team:Hawaii/Notebook/2008-08-14

From 2008.igem.org

(Difference between revisions)
(Quote of the Day)
m (Restriction Digest)
 
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::* Also ran J33207 PCR product on same gel
::* Also ran J33207 PCR product on same gel
::* Excised J33207 and GFPf bands from gel
::* 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.]]
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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.]]
::* J33207, J04430, BB-pRL1383a with EcoRI and PstI
::* J33207, J04430, BB-pRL1383a with EcoRI and PstI
::* pRL1383a, J33207 with HindIII and BamHI (Roche, from SC)
::* pRL1383a, J33207 with HindIII and BamHI (Roche, from SC)
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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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===Gel extraction===
===Gel extraction===

Latest revision as of 19:16, 15 August 2008

Projects Events Resources
Sponsors Experiments Milestones Protocols
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


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