Team:Hawaii/Notebook/2008-07-29

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===Construction of BioBricks===
===Construction of BioBricks===
:<strong>Grace</strong>
:<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.]]
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:* Purified RE digests on EtBr stained 3% agarose gel; extracted desired bands
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:* Purified RE digests on EtBr stained 1.2% agarose gel
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:* Ligated pRL1383a with ccdB, nir with B0030, and GFP and GFPf with B0024
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::* Extracted bands for B0030 and B0024
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::* Other lanes FAILED!
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===Transformation of constructs in DB3.1===
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::* SYBR Safe doesn't show up well, even under UV (arg...)
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:<strong>Grace</strong>
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===Direct Band Extraction Method===
===Direct Band Extraction Method===
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:<strong>Norman</strong>
:* make two comb gels
:* make two comb gels
:* run test UPA band along with ladder
:* run test UPA band along with ladder
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:* no more gel cutting???
:* no more gel cutting???
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== Drylab Work ==
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===PCR amplification of pRL1383a parts===
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:<strong>Margaret</strong>
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:* Made a reaction buffer containing {38.5ul, 20ul accusure, 10ul dNTPs, & 5ul 5XBuffer}. This should be good for 20 50ul reactions.
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===Name of Task===
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:*Amplified with Accusure: aadA ([http://partsregistry.org/Part:BBa_J23012 BBa_J23012]), aadaA (pRL1383a), rep region, oriV
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:<strong> name of person/people who performed the task</strong>
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:*Amplified with Green Taq: rep region
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:* Summary of task and what was done. Link to experiment for detailed notes if necessary.
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:*On [[Team:Hawaii/Notebook/2008-07-30|7-30]] I ran a gel.
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:* e.g. read through papers, worked on proposal, etc.
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===Media===
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:<strong>Margaret</strong>
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:*started to make LB amp100 plates, but autoclave room is closed. Remember to do it tomorrow.
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===Plasmid prep===
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:<strong>Krystle</strong>
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:* Innoculated LB+amp with pRL1383a harboring E. coli (from cryostock)
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===PCR===
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:<strong>Krystle</strong>
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:* PCR reactions to isolate GFP, GFPf, nir from plasmid
= Discussion =
= Discussion =
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''' SYBR Safe'''
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:* 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 =
= Quote of the Day =

Latest revision as of 09:40, 31 July 2008

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Notebook (t) Meetings (t)

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 1.2% agarose gel
  • Extracted bands for B0030 and B0024
  • Other lanes FAILED!
  • SYBR Safe doesn't show up well, even under UV (arg...)

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.

Plasmid prep

Krystle
  • Innoculated LB+amp with pRL1383a harboring E. coli (from cryostock)

PCR

Krystle
  • PCR reactions to isolate GFP, GFPf, nir from plasmid

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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