Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/07/03: 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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== | ==07/3/2015== | ||
<font color = purple> | <font color = purple> | ||
Ligate modular receiver vector (MRV) + RhlR, LuxR, and EsaR. < | <h4 font color = purple> Ligate modular receiver vector (MRV) + RhlR, LuxR, and EsaR. </h4> | ||
<b>1. Digest 1ug MRV </b> | <b>1. Digest 1ug MRV </b> | ||
{| {{table}} | {| {{table}} | ||
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|} | |} | ||
Incubate at 37°C for 15 minutes. <br> | :Incubate at 37°C for 15 minutes. <br> | ||
Heat inactivate at | :Heat inactivate at | ||
:*80°C for 5 minutes | |||
:*65°C for 20 minutes | |||
<b> 2. Digest PCR amplified and purified, regulators w/ E+X. </b> | <b> 2. Digest PCR amplified and purified, regulators w/ E+X. </b> | ||
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<b>3. Ligate 3:1 ratio of insert:bb </b> | <b>3. Ligate 3:1 ratio of insert:bb </b> | ||
Amounts of DNA used were based on vector/insert size. <br> | :Amounts of DNA used were based on vector/insert size. <br> | ||
Vector is ~3,200bp. Transcription factor inserts are ~851bp. <br> | :Vector is ~3,200bp. Transcription factor inserts are ~851bp. <br> | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | | align="center" style="background:#f0f0f0;"|'''Reagent''' | ||
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<br> | <br> | ||
<b> 4. Transformation with DH5αT cells </b> <br> | <b> 4. Transformation with DH5αT cells </b> <br> | ||
10uL of each ligation reaction were used in the transformation. | :10uL of each ligation reaction were used in the transformation. | ||
---- | ---- | ||
PCR amplify inducible promoters for: Aub, Bta, Cer, and Exp. (Bta, Cer, and Exp seemed to be running low; unsure if these will work | PCR amplify inducible promoters for: <br> | ||
:Aub, Bta, Cer, and Exp. | |||
:(Bta, Cer, and Exp seemed to be running low; unsure if these will work) | |||
Latest revision as of 01:02, 27 September 2017
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07/3/2015
Ligate modular receiver vector (MRV) + RhlR, LuxR, and EsaR.1. Digest 1ug MRV
2. Digest PCR amplified and purified, regulators w/ E+X.
3. Ligate 3:1 ratio of insert:bb
|