Team:Hawaii/Notebook/2008-08- 5

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(→Construction of p+r and g+t, otra vez)
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:* Jabbed nir and GFPf cryostocks and put into LB+amp<sub>100</sub>  
:* Jabbed nir and GFPf cryostocks and put into LB+amp<sub>100</sub>  
::* Also need plasmid prep of this to submit to registry
::* Also need plasmid prep of this to submit to registry
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::* Since both nir PCR RE'd didn't show distinct bands, perhaps we should RE'd nir plasmid to make nir+rbs (if today's p+r doesn't work)
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::* Since both nir PCR RE'ds didn't show distinct bands, perhaps we should RE'd nir plasmid to make nir+rbs (if today's p+r doesn't work)
===Construction of p+r and g+t, otra vez===
===Construction of p+r and g+t, otra vez===

Revision as of 04:00, 6 August 2008

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Things we did today

Wetlab work

Transformation

Grace
  • Transformed 50 μl DB3.1 competent cells with 5 μl ligation reactions of p+r or g+t
  • Incubated on ice 17 minutes after adding DNA

PCR contamination test (cont. from 8/2 and 8/4)

Grace
  • Ran an EtBr stained 2.5% gel at 95V for 2 hours
  • Why aren't I getting consistent results for any of the lanes?
  • GP says not to worry about contaminants -- they're there, let's move on with life
  • On 8/5 gel, potentially see desired 1.1kb band
  • Cut out of gel, use as template for another PCR to verify it's what we want --OR-- plasmid prep colony (decided to plasmid prep since we need to send in a plasmid prep to partsregistry eventually)

Inoculated for plasmid prep tomorrow

Grace
  • Colony #1 of pRL1383a 1:2 into LB+sp100
  • Jabbed nir and GFPf cryostocks and put into LB+amp100
  • Also need plasmid prep of this to submit to registry
  • Since both nir PCR RE'ds didn't show distinct bands, perhaps we should RE'd nir plasmid to make nir+rbs (if today's p+r doesn't work)

Construction of p+r and g+t, otra vez

Grace
  • 10 μl PCR reactions for nir, slr1, slr2, pilA, J33207 (in case transformation doesn't work)
  • RE digest of PCR products (no gel purification; used 3 μl PCR reactions)
  • Digested nir and J33207 with EcoRI and SpeI
  • Digested slr1, slr2, pilA with XbaI and PstI
  • Ran 20 μl of digests on 2% agarose gel stained with EtBr for 2.5 hours
  • Extracted slr1, slr2, pilA
  • J33207 didn't cut? Bands at 830bp and 2.5kb
  • nir was a smear between 110-140bp; didn't cut because desired band (~90bp) not present

Re-streak

Margaret
  • Restreaked stuff from yesterday, including pSMC121 which was a lawn, the lac promoter (on fresh plate containing Kan50 & 1mM IPTG), and DB3.1 stock on fresh Sm50 plate.

Gels

Margaret
  • ran gels for yesterday's PCR and Quantification experiments.

PCR

Margaret
  • testing PCR conditions for P1 lytic region and aadA (registry and pRL1383a)

Drylab Work

Sequencing

Grace
  • Reviewed sequencing results from 7/21, emailed CORE Hawaii with concerns
  • Mislabeled samples
  • Poor quality reads

Discussion

  • Looks like our TriDye 2-log ladder has gone bad (500bp band splits into two bands). Norman will aliquot out new ladder.

Quote of the Day

History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson


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