Team:Yale/Our Project/Notebook/Week 4

From 2010.igem.org

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Ran PCR products on an 0.8% agarose gel with 10 uL of ethidium bromide.  Loaded 1 uL of each sample with 9 uL of water and 2 uL of loading buffer.  Ran gel until done at 60 V, but had difficulty imaging so left aside to deal visualize the next day, unaware that it would spread. <br/>
Ran PCR products on an 0.8% agarose gel with 10 uL of ethidium bromide.  Loaded 1 uL of each sample with 9 uL of water and 2 uL of loading buffer.  Ran gel until done at 60 V, but had difficulty imaging so left aside to deal visualize the next day, unaware that it would spread. <br/>
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<i>This day's labwork is also recorded on pages 23-28 of the hard copy lab notebook </i>
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Tuesday 6/29--Gel extraction of PCR amplified phs, TSI agar slant assay for hydrogen sulfate production
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, & pre-ligation double digestion
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<h4>PCR amplification of phs </h4>
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<li> Because the gel run the previous day was no longer good, had to run a second 0.8% agarose gel of the PCR products to determine the success of the PCR reaction. Below is the image of the gel, with a 1 kb ladder in the leftmost lane, phsC in next lane, phsAB in lane 3, and phsABC in lane 4. </li>
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The phsC fragment seems to have been amplified with decent success, but phsABC and phAB are only present in trace amounts, so the thermocycler protocol will have to be tweaked. <br/>
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<li> In the mean time, gel extract the phsC from lane 2 and the phsABC from lane 4 using the microcentrifuge variant of the  <a href="http://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract">standard gel extraction protocol </a> </li>
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<h4> TSI Agar Slant Assay </h4>
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<li> Want to confirm that IPTG will induce H<sub>2</sub>S production in pSB74 transformants, so inoculate 5 mL Amp/LB liquid cultures of DH5alpha and LE392 transformed with pSB74 and grow them up on shaker at 37˚C for use in TSI agar assay. </li>
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<li> The slants don't contain IPTG, so must inject IPTG with the transformants into the slants. Took 200 uL of each cell solution and added to it 50 uL of 1 M IPTG, then injected the resulting mixture into a slant.  Left the slants to sit overnight in the fume hood </li>
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<h4> Double digestion in preparation for ligation </h4>
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In order to create the desired Biobricks, must insert the phsABC operon into terminator B0015 and put the constitutive promoter J23114 into the generator P0312.  Following the Biobrick combination protocol, we then must digest phsABC and J23114 with EcoRI and SpeI and digest B0015 and P0312 with EcoRI and XbaI.  The former can be done as a double digest, but the latter must be done sequentially because the enzymes require different buffers.<br/>
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Set up the following 50 uL digestions:<br/>
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<b>Digest of phsABC</b>--5 uL EcoRI buffer, 0.5 uL 100x BSA, 40 uL phsABC fragment (from gel extraction), 1.8 uL EcoRI, 1.8 uL SpeI, 0.9 uL water <br/>
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<b>Digest of J23114</b>--5 uL EcoRI buffer, 0.5 uL 100x BSA, 8 uL miniprepped J23114 (1 ug DNA), 1.8 uL EcoRI , 1.8 uL SpeI, 32.9 uL water <br/>
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<b>Digest of B0015</b>--5 uL EcoRI buffer,  12 uL miniprepped B0015 (1 ug DNA), 3.6 uL EcoRI , 29.4 uL water <br/>
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<b>Digest of P0312</b>--5 uL EcoRI buffer,  9 uL miniprepped P0312 (1 ug DNA), 3.6 uL EcoRI , 32.4 uL water <br/>
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Let each digestion run for two hours at 37˚C, then ran the result through the microcentrifuge version of the <a href="http://2010.igem.org/Team:Yale/Our_Project/Protocols/PCR_purify">PCR purification protocol.</a>
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<i>This day's labwork is also recorded on pages 28-31 of the hard copy lab notebook </i>
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Revision as of 10:30, 27 October 2010

iGEM Yale

lab notebook: week 4 (6/28 -7/4)

  • Monday 6/28--PCR amplification of thiosulfate reductase operon phsABC
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  • Tuesday 6/29--Gel extraction of PCR amplified phs, TSI agar slant assay for hydrogen sulfate production , & pre-ligation double digestion
  • See more/less
  • Wednesday short content
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  • Thursday short content
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  • Fridays short content
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  • Saturday short content
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  • Sunday short content
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