Team:Hawaii/Notebook/2008-08- 8

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===Prep for sequencing===
===Prep for sequencing===
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[[Image:080808PCR.jpg|right|thumb|200px|EtBr stained 4% agarose gel ran at 60V for 100 min. Twenty-five microliters of the PCR reactions were loaded into each well.]]
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[[Image:080808seqPCR.jpg|right|thumb|400px|EtBr stained 4% agarose gel ran at 60V for 100 min. Twenty-five microliters of the PCR reactions were loaded into each well.]]
:<strong>Grace</strong>
:<strong>Grace</strong>
:* 25 &mu;l PCR reactions of nir+rbs, I14032+rbs, slr1, slr2, BB-pRL1383a
:* 25 &mu;l PCR reactions of nir+rbs, I14032+rbs, slr1, slr2, BB-pRL1383a
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===Construct p+r+g and p+r+s===
===Construct p+r+g and p+r+s===
:<strong>Krystle</strong>
:<strong>Krystle</strong>
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[[Image:080808resdig.jpg|right|thumb|200px|EtBr stained 2% agarose gel ran at 60V for 60 min. Forty microliters of each digest were loaded.]]
:* Restriction Digest
:* Restriction Digest
:**nir+B0030, I14032+B0030, J33207 digested with SpeI and PstI
:**nir+B0030, I14032+B0030, J33207 digested with SpeI and PstI
:**gfp, gfp''fusion'', and B0015 digested with XbaI and PstI
:**gfp, gfp''fusion'', and B0015 digested with XbaI and PstI
:* Gel Purified restriction digest
:* Gel Purified restriction digest
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::: 2% agarose gel ran at 60 volts for 1.5 hours
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:** 2% agarose gel ran at 60 volts for 1.5 hours
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:** 40ul of the total rxn loaded into each well
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::: 40ul of the total restriction digest loaded into each well
-
:** gfp, gfp''fusion'', I14032+B0030, nir+B0030 extracted using Qiagen MiniElute tubes
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::* gfp''fusion'' cut out from gel
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:** B0015 and J33207 extracted using Qiagen cDNA purification tube
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= Discussion =
= Discussion =

Latest revision as of 19:16, 9 August 2008

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

Things we did today

Wetlab work

Restreaked nir+rbs and I14032+rbs constructs

Grace

Plasmid prep (finished up)

Grace
  • Resuspended plasmid preps in 50 μl TE buffer
  • Determined DNA concentrations of plasmids
Plasmid DNA Concentration
nir 496.3 ng/μl
GFPf 484.8 ng/μl
BB-pRL1383a 508.0 ng/μl

Prep for sequencing

EtBr stained 4% agarose gel ran at 60V for 100 min. Twenty-five microliters of the PCR reactions were loaded into each well.
Grace
  • 25 μl PCR reactions of nir+rbs, I14032+rbs, slr1, slr2, BB-pRL1383a
  • Colony PCRs seem to indicate success
  • 25 μl PCR reactions of GFP+tt, GFPf+tt, J33207+tt
  • Colony PCR indicates no ligation. Picked new colony, sequence to confirm failure.
  • PCR of nir, B0015, B0030, B0034 for resequencing (bad reads last time)
  • Gel purified all PCR rxns (we still have a problem with contaminant DNA/shadow bands) and desired bands were extracted from gel
  • 2% agarose gel ran at 60v for 2 hours
  • Determined DNA concentrations via nanodrop spectrometer
  • Prepared samples and sent to CORE Hawaii for sequencing

Construct p+r+g and p+r+s

Krystle
File:080808resdig.jpg
EtBr stained 2% agarose gel ran at 60V for 60 min. Forty microliters of each digest were loaded.
  • Restriction Digest
    • nir+B0030, I14032+B0030, J33207 digested with SpeI and PstI
    • gfp, gfpfusion, and B0015 digested with XbaI and PstI
  • Gel Purified restriction digest
    • 2% agarose gel ran at 60 volts for 1.5 hours
40ul of the total restriction digest loaded into each well
  • gfpfusion cut out from gel

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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