Team:Lambert GA/labnotebook

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                 <li><a href="https://2013hs.igem.org/Team:Lambert_GA/activities" accesskey="6" title="">Activities</a></li>               
                 <li><a href="https://2013hs.igem.org/Team:Lambert_GA/activities" accesskey="6" title="">Activities</a></li>               
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<center><b><u><h1>Lab Notebook</h1></u></b></center>
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<font size=4><center><b><u><h1>Lab Notebook</h1></u></b></center></font>
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<td>June 11th</td>
<td>June 11th</td>
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<td>We ran a diagnostic gel, which showed the best temperature for PCR was 46.1°C and 53.3°C. On the gel we got 500 base pairs, which is the expected length of our primers and part. We used QIAprep for PCR cleanup.</td>
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<td>We ran a gradient PCR with HybB and R0011 as control. diagnostic gel showed the best temperature for PCR was 44.6°C and 53.3°C. On the gel we had bands around 400 base pairs, which is the expected length of our primers and part. We used QIAprep for PCR cleanup. We decided to combine 4 PCR reactions and purify to use in digestions and ligations. After purification HybB is 27.4ng/ul and control was 9.9 ng/ul.
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<td>June 12th</td>
<td>June 12th</td>
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<td>We met with Georgia Tech iGem team and heard their presentation and discussed cooperation and outreach. We did a double digest with EcoRl remix and PST1. We blasted all of our parts to check all of the DNA sequences against the parts registry. We also worked on the wiki and the poster.</td>
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<td>We met with Georgia Tech iGem team and heard their presentation and discussed cooperation and outreach. We did a double digest on HybB, PsB1C3, and control R0011. We used EcoRl remix and PST1. We blasted all of our parts to check all of the DNA sequences against the parts registry. We ligated HybB and PSB1C3, and a Control (R0011 and PSB1C3). In order to keep the backbones from reclosing we used Antarctic phosphatase to eliminate the phosphates on the ends.  We also ligated 3 ratios of backbone to part. 1:1, 1:2 and 1:3. Ligase came to room temperature.</td>
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<td>June 13th</td>
<td>June 13th</td>
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<td>We repeated the double digest. We used Antarctic phosphatase to cut off the extra phosphates on the 5 prime ends of the backbone. We ligated the HybB promoter and the backbone and we included a control of just the backbone. Then we transformed the ligation mixtures of today and yesterday resulting in eight plates.</td>
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<td>We repeated the double digest because the ligase was suspect. We used Antarctic phosphatase to cut off the extra phosphates on the 5 prime ends of the backbone. We ligated the HybB promoter and the backbone and we included a control of just the backbone. Then we transformed the ligation mixtures from 6/12, 6/13, and a control. Our control was the backbone and R0011.</td>
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<td>June 14th</td>
<td>June 14th</td>
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<td>We recorded the results from yesterday, then we did colony PCR on the colonies, choosing the ligation mixture that was a 2:1 ratio of HybB to backbone. We did a diagnostic gel with 16 colonies and analyzed our results. Unfortunately, our colony PCR failed. We do not know the reason for the failure, but we suspect that  
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<td>We recorded the results of the plates. There were no colonies from the 6/12 ligations. Pictures of the 6/13 ligations have been included.
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<center><b>1:1 Ratio</b>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<b>1:2 Ratio</b>
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<center>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<b>1:3 Ratio</b>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<b>Antarctic Phosphatase (Just backbone)</b>
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<br><center><b>Control R0011</b></center>
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We did colony PCR using the HybB promers for 16 colonies, choosing the ligation mixture that was a 2:1 ratio of HybB to backbone. We set the thermocycler to 30 cycles annealing 50 degrees. Our elongation was 30 seconds. We did a diagnostic gel with 16 colonies and analyzed our results. Unfortunately, our colony PCR failed. We do not know the reason for the failure, but we suspect that
<b>a)</b>we did not put the colonies in the thermocycler for long enough, or
<b>a)</b>we did not put the colonies in the thermocycler for long enough, or
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<td>June 17th</td>
<td>June 17th</td>
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<td>We repeated the colony PCR.</td>
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<td>We repeated the colony PCR with 16 new colonies and a control. We set the elongation step to 45 seconds, thinking that the sequences may not have had time to finish.  All other steps remained the same.  Again, there were no expected or unexpected bands. Then, we inoculated liquid cultures with 5 different colonies from the plates of ligations.
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June 18th
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Then we miniprepped the liquid cultures. We found concentrations of DNA range from 79.5ng/ul to 114.5ng/ul. Then they were digested with EcoR 37C for 15 minutes. We ran diagnostic gel on the digests.  We were expecting bands at 2070 bp for just PSB1C3 and 2463 for Colonies which contained HybB promoter sequence.  We also ran them with a control of R0011.
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June 19th</td>
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<td> Amplification of HybB from DH10Beta and control.  We used different concentrations of DH10 from 2 sources (ÜberDan's and ours), along with a control of R0011 in PSB1c3. The amplification failed, and the gel showed no bands.  The pipetting of Taq was suspected to have caused the failure
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Latest revision as of 03:49, 22 June 2013