Team:Yale/Our Project/Notebook/Week 4

From 2010.igem.org

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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/>
<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/>
<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/>
<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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<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/><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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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><br/>
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Wednesday short content
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Wednesday 6/30--Troubleshooting PCR amplification of thiosulfate reductase gene & continued work toward ligation
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<b>TSI Agar slant </b></br>
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<li>No visible color change, and decide that it is safe to accelerate growth by moving slants to the 37˚C incubator as research fails to turn up any safety concerns regarding the hydrogen sulfide emitted in this kind of assay. </li>
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<b>PCR efforts continue </b><br/>
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<li>Redid 6/29 PCR of phsABC and phsAB, but this time ran two trials of each, with and without 3% DMSO, and changed the thermocycler protocol so that the annealing temperature was 50˚C rather than 55˚C, calling the new protocol "phs50."</li>
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<li> After running the protocol, found that PCR tubes had popped open and contents had evaporated, so set up PCR a second time to run overnight.</li>
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<h4>Vector digestion & 1st ligation attempt</h4>
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<li> Ran the second portion of the B0015 and P0312 sequential digests, with reaction contents of each as follows: 5 uL NEB Buffer 4, 0.5 uL 100x BSA, 3.6 uL XbaI, 40 uL DNA from PCR purification protocol, and 0.9 uL water.  Let the reactions run for 2.5 hours at 37˚, but after 1.5 hours added to each reaction 1 uL of Calf Intestinal Phosphatase (CIP) to cleave terminal phosphates and discourage self-ligation in the next step. </li>
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<b>Ligation of phsABC and B0015</b> <br/>
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By this point the DNA concentrations of the digestion reactions are dismally low (around 2 ng/uL for B0015 and phsABC), but will try a ligation anyways while at the same time preparing for a possible redo. Set up digestion reactions of two sizes, 10 uL and 20 uL, with a control reaction for each in which there is no insert.<br/>
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<b> Reaction 1--small control</b> 1 uL ligase buffer, 0.5 uL T4 ligase, 4.25 uL B0015, 4.25 uL water<br/>
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<b> Reaction 2--small 1:1 insert:vector</b>1 uL ligase buffer, 0.5 uL T4 ligase, 4.25 uL B0015, 4.25 uL phsABC<br/>
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<b> Reaction 3--small 2:1 insert:vector</b>1 uL ligase buffer, 0.5 uL T4 ligase, 2.5 uL B0015, 6 uL phsABC<br/>
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<b> Reaction 4--large control</b>2 uL ligase buffer, 1 uL T4 ligase, 8.5 uL B0015, 8.5 uL water<br/>
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<b> Reaction 5--large 1:1 insert:vector</b>2 uL ligase buffer, 1 uL T4 ligase, 8.5 uL B0015, 8.5 uL phsABC<br/>
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Insert to vector ratios are rough and based on the fact that the two are at similar concentrations and are of similar sizes. <br/>
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Each reaction was let to run 20 minutes at room temperature before being transformed into Xl1-Blue cells according to the standard <a href="http://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> transformation protocol </a> and then plated on ampicillin LB plates and let to grow overnight.<br/>
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<b> Cultures for miniprep & glycerol stock redo </b> <br/>
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In preparation for potential ligation failure, once again inoculated liquid cultures of Amp LB  for miniprep with pSB74,P0312,R0011, B0015, and J23114 transformants, making two 5 mL cultures for each plasmid.  When OD reached 0.6, once again made glycerol stocks from 0.5 mL of each solution and 0.5 mL of filter-sterilized 50% glycerol.  This time flash-froze the stocks immediately, rather than waiting overnight as.  <br/>
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<i> This day's labwork is also recorded on pages 32-36 of the hard copy lab notebook. </i>
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Revision as of 11:08, 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 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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