Team:Hawaii/Notebook/2008-08- 8

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(Difference between revisions)
(New page: {{Team:Hawaii/Header}} = Things we did today = == Wetlab work == ===Restreaked nir+rbs and I14032+rbs constructs=== :<strong> Grace</strong> ===Prep for sequencing=== [[Image:080808PCR.j...)
(→Construct p+r+g and p+r+s)
 
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===Restreaked nir+rbs and I14032+rbs constructs===
===Restreaked nir+rbs and I14032+rbs constructs===
:<strong> Grace</strong>
:<strong> Grace</strong>
 +
 +
===Plasmid prep (finished up)===
 +
:<strong>Grace</strong>
 +
 +
:* Resuspended plasmid preps in 50 &mu;l TE buffer
 +
:* Determined DNA concentrations of plasmids
 +
{|class=wikitable border=1 align=center
 +
!Plasmid
 +
! DNA Concentration
 +
|-
 +
|align=center |nir
 +
|align=center |496.3 ng/&mu;l
 +
|-
 +
|align=center |GFPf
 +
|align=center |484.8 ng/&mu;l
 +
|-
 +
|align=center |BB-pRL1383a
 +
|align=center |508.0 ng/&mu;l
 +
|}
===Prep for sequencing===
===Prep for sequencing===
-
[[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.]]
+
[[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
Line 13: Line 32:
:* 25 &mu;l PCR reactions of GFP+tt, GFPf+tt, J33207+tt
:* 25 &mu;l PCR reactions of GFP+tt, GFPf+tt, J33207+tt
::* Colony PCR indicates no ligation. Picked new colony, sequence to confirm failure.
::* 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
:* 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
:* Determined DNA concentrations via nanodrop spectrometer
:* Prepared samples and sent to CORE Hawaii for sequencing
:* Prepared samples and sent to CORE Hawaii for sequencing
 +
===Construct p+r+g and p+r+s===
 +
:<strong>Krystle</strong>
 +
[[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
 +
:**nir+B0030, I14032+B0030, J33207 digested with SpeI and PstI
 +
:**gfp, gfp''fusion'', 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
 +
::* gfp''fusion'' cut out from gel
= 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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