Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/07/03: Difference between revisions
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<br> | <br> | ||
Amount of DNA used for each reaction | Amount of DNA used for each reaction w/r to their concentrations: | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''RhlR''' | | align="center" style="background:#f0f0f0;"|'''RhlR''' | ||
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|- | |- | ||
| 3.7||3.6||5.9 | | 3.7||3.6||5.9 | ||
|} | |||
<br><br> | |||
<b>3. Ligate 3:1 ratio of insert:bb </b> | |||
Amounts of DNA used were based on vector/insert size. <br> | |||
Vector is ~3,200bp. Transcription factor inserts are ~851bp. <br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (uL)''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| ||EsaR||RhlR||LuxR ||Vector-only | |||
|- | |||
| Insert DNA (40ng)||6uL ||6uL ||6uL||0uL | |||
|- | |||
| Vector DNA (50ng)||1.52uL||1.52uL||1.52uL||1.52uL | |||
|- | |||
| 10x Buffer||2uL||2uL||2uL||2uL | |||
|- | |||
| Ligase||1uL||1uL||1uL||1uL | |||
|- | |||
| Water ||9.48uL||9.48uL||9.48uL||15.48uL | |||
|- | |||
| Total ||20uL||20uL||20uL||20uL | |||
|} | |} | ||
Revision as of 17:42, 3 July 2015
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06/3/2015
Performed ligations of modular receiver vector + RhlR, LuxR, and EsaR regulator ORFs.
Incubate at 37°C for 15 minutes. 2. Digest PCR amplified, then purified, regulator ORFs with EcoRI and XbaI.
3. Ligate 3:1 ratio of insert:bb
Amounts of DNA used were based on vector/insert size.
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