Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/03/31: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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Today Ryan double | Today Ryan helped me double digest more backbone with EcoRI and XbaI for the second ligation. <br> | ||
I PCR purified the reactions and eluted at 45uL with warm water. <br> <br> | I PCR purified the reactions and eluted at 45uL with warm water. <br> <br> | ||
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| align="center" style="background:#f0f0f0;"|'''Water''' | | align="center" style="background:#f0f0f0;"|'''Water''' | ||
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| | | AubI||29||2.16||4.17||10.67 | ||
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| BjaI||30||2.08||4.17||10.75 | | BjaI||30||2.08||4.17||10.75 | ||
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| SinI||47||1.33||4.17||11.5 | | SinI||47||1.33||4.17||11.5 | ||
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| Bb only ||39|| | | Bb only ||39||0||4.17||12.83 | ||
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All backbone came from the 12ng/uL PCR-purified product. <br> <br> | |||
2uL of T4 10x ligase buffer and 1uL of ligase were added to each reaction as well. The total volume is 20uL. <Br> | 2uL of T4 10x ligase buffer and 1uL of ligase were added to each reaction as well. The total volume is 20uL. <Br> | ||
I incubated reactions at room temperature for 10 minutes. Then, I followed with a long transformation. <br><Br> | I incubated reactions at room temperature for 10 minutes. Then, I followed with a long transformation. <br><Br> |
Latest revision as of 00:52, 27 September 2017
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03/31/2015
Today Ryan helped me double digest more backbone with EcoRI and XbaI for the second ligation. After purification, I pipetted the following contents into PCR tubes:
2uL of T4 10x ligase buffer and 1uL of ligase were added to each reaction as well. The total volume is 20uL. I pipetted all contents (20uL) of each tube into a new 2.0mL tube, then added 50uL of DH5αT cells to each. I also created a negative control with just cells and 20uL water. I had a total of 11 tubes. I allowed them to incubate on ice for 30 minutes, then heat shocked at 42°C for 35 seconds, and incubated on ice for another 2 minutes. Following this, I pipetted 750mL of SOC into each tube. Then I taped the tubes in an empty agar plate and let it shake in the shaker for 1 hour. After the hour, I spun the tubes down, removed 500uL of SOC, resuspended the fluids and pipetted the contents (~300uL) into plates that have been labeled and warmed. I used beads to mix the contents in the plate and then placed them upside down in the 37°C incubator overnight. |