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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<font size=4><center><b><u><h1>Lab Notebook</h1></u></b></center></font>
<font size=4><center><b><u><h1>Lab Notebook</h1></u></b></center></font>
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<th>Date</th>
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<td>June 11th</td>
<td>June 11th</td>
<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.
<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 14th</td>
<td>June 14th</td>
<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.
<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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1:1 Ratio
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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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1:2 Ratio
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1:3 Ratio
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Antarctic Phosphatase (Just backbone)
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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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Control R0011
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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
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
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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