Team:Hawaii/Notebook/2008-07-29

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===Construction of BioBricks===
===Construction of BioBricks===
:<strong>Grace</strong>
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[[Image:072908REdigests.jpg|right|200px|thumb|EtBr stained 1.2% agarose gel. Thirty microliters of each RE reaction and 10 microliters of ccdB were loaded into wells. Gel ran at 60V for 2 hours.]]
:* Purified RE digests on EtBr stained 3% agarose gel
:* Purified RE digests on EtBr stained 3% agarose gel
::* Extracted bands for B0030 and B0024
::* Extracted bands for B0030 and B0024

Revision as of 09:37, 31 July 2008

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Things we did today

Wetlab work

Gel purification of ccdB PCR product

Grace

RE digests

Grace
  • Digested pRL1383a (from last night) with BamHI
  • Digested B0030 and B0024 (from last night) with EcoRI
  • Sequentially digested gel purified ccdB with HindIII and BamHI

Construction of BioBricks

Grace
EtBr stained 1.2% agarose gel. Thirty microliters of each RE reaction and 10 microliters of ccdB were loaded into wells. Gel ran at 60V for 2 hours.
  • Purified RE digests on EtBr stained 3% agarose gel
  • Extracted bands for B0030 and B0024
  • Other lanes FAILED!

Direct Band Extraction Method

Norman
  • make two comb gels
  • run test UPA band along with ladder
  • attempt to pipette out DNA as it runs into the second well
  • no more gel cutting???

PCR amplification of pRL1383a parts

Margaret
  • Made a reaction buffer containing {38.5ul, 20ul accusure, 10ul dNTPs, & 5ul 5XBuffer}. This should be good for 20 50ul reactions.
  • Amplified with Accusure: aadA ([http://partsregistry.org/Part:BBa_J23012 BBa_J23012]), aadaA (pRL1383a), rep region, oriV
  • Amplified with Green Taq: rep region
  • On 7-30 I ran a gel.

Media

Margaret
  • started to make LB amp100 plates, but autoclave room is closed. Remember to do it tomorrow.

Discussion

SYBR Safe

  • Blue light still doesn't illuminate the bands well. No bands (not even the ladder) are visible. Resolution *much* better with short wave UV. EtBr better bet for now?

Restriction enzymes

  • HindIII and BamHI in the lab -20C freezer (in box labeled "TKW Restriction Enzymes") fail. They're really old and don't appear to cut well anymore. Digestion of pRL1383a resulted in a long smear.

Quote of the Day

LOL! http://www.youtube.com/watch?v=J0s0Y3-BCaw


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