Team:Hawaii/Notebook/2008-07-30

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Things we did today

Wetlab work

Restriction Digest

Grace
EtBr stained 1.2% agarose gel. Twenty microliters of the restriction digest reactions were loaded into each well. Gel ran at 59V for 1.5 hours.
  • Digested 5 μl GFP, GFPf, and nir PCR products with EcoRI and SpeI
  • Sequentially digested 10 μl pRL1383a and 5 μl J33207 PCR product with BamHI and HindIII
  • Used Roche enzymes and Roche buffer M (buffer B also works) from SC
  • Ran digests on gel
  • pRL1383a and J33207 bands good (excised)
  • GFP, GFPf, and nir bands still too big
Krystle
  • Digested 10 ul each of GFP, GFPf, and nir PCR products from Grace's PCR on 07/30/08 with EcoRI and SpeI

PCR

Grace and Krystle
  • Redid yesterday's PCR of GFP, GFPf, and nir
  • Reran on gel, producing almost identical results. Bands were excised.

Gel Purification

Grace
  • Purified B0030 and B0024 from gel
  • B0030 = 21.9 ng/μl
  • B0024 = 5.7 ng/μl
Krystle
  • Purified restricted pRL1383 and J33207 from gel
  • Purified Grace's PCR products of GFP, GFPfusion, and nir from gel
  • Why are bands still ~1kb and ~1.2kb?

Plasmid Prep

Krystle
  • Maxiprep of pRL1383a

PCR amplification of pRL1383a

Margaret
PCR amplification of oriV, aada, rep.
  • ran a gel from yesterday's PCR amplification of oriV, aadA, and rep regions.

Mini-Prep

Margaret

Colony PCR

Margaret
A colony PCR verification of BBa_I14032, a lac Promoter and the oriT construct.
  • verify the insert of the oriT construct and BBa_I14032
  • Results:
  • BBa_I14032 should be 334bp, the band under 0.4kb indicating a verification of this size of insert.
  • oriT should be about 415bp(+BioBrick ends), the band is around 0.4kb so is in approximately the right place.
  • Discussion: The a plasmid prep and glycerol stock of both will be made. I need to verify the oriT part through sequencing.

Discussion

The gel purification kit is almost out of PE. If we need more before our order comes in, there is PE in the Qiaprep Spin Miniprep kit (just remember to add EtOH before use).

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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