Team:Yale/Our Project/Notebook/Week 4

From 2010.igem.org

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Saturday 7/3--More attempts to put a terminator on thiosulfate reductase and a promoter on lacI
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<h4>Ligation Attempt #2 continued</h4>
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<li>Following digestion and purification, took concentrations of digestion products, which were still miserably low (phsABC: 3.2 ng/uL, P0312: 16.88 ng/uL, J23114: 23.98 ng/uL, B0015: 13.24 ng/uL) and set-up ligation attempt #2. For each of the two ligations (phsABC into B0015 and J23114 into P0312) four different 20 uL reactions were run: a control ligation with vector, ligase and buffer but no insert, a reaction with a 2:1 stoichiometric ratio of insert to vector, a reaction with a 3:1 insert to vector ratio, and a reaction with a 5:1 insert to vector ratio. All reactions used 1 uL NEB T4 ligase and 2 uL of accompanying 10x buffer and were run for 15 minutes at room temperature. Following this the (theoretically) ligated plasmids were transformed into competent XL1-Blue cells according to the <a href="http://2010.igem.org/Team:Yale/Our_Project/Protocols"> standard transformation protocol </a> and plated onto Amp plates</li>
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<li>Index plates from ligation attempt #1 on 7/1 belatedly showed growth. </li>
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<i>This day's activities are also recorded on page 45 of the hard copy lab notebook </i>
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Revision as of 11:50, 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 sulfide production, & pre-ligation double digestion
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  • Wednesday 6/30--Troubleshooting PCR amplification of thiosulfate reductase gene & continued work toward ligation
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  • Thursday 7/1--Smelly bacteria (yay!) & ongoing ligation efforts
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  • Fridays 7/2--Confirmation of hydrogen sulfide production plus more ligation work
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  • Saturday 7/3--More attempts to put a terminator on thiosulfate reductase and a promoter on lacI
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  • Sunday short content
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