Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/07/03: Difference between revisions
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<font color = purple> | <font color = purple> | ||
<h4> Ligate modular receiver vector (MRV) + RhlR, LuxR, and EsaR. </h4> | <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) | |||
Revision as of 17:50, 3 July 2015
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
06/3/2015
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
|