Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/04/29: 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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<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
==mm/dd/yyyy== | ==mm/dd/yyyy== | ||
Ran PCR products from yesterday on a gel. 5ul of reactions 2-9, all 30 of 1 so I can gel extract because there should be two bands and I only want one. | |||
<br> | |||
DpnI digest PCR reactions 2-7 and 9 with positive and negative controls (didn't run reaction 8 because I checked the gel early and saw that there was no DNA there). | |||
<br> | |||
Controls | |||
Positive control was a miniprepped plasmid at 100ng/ul concentration. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| Thing||Positive ctrl||Negative ctrl | |||
|- | |||
| H2O||12||12 | |||
|- | |||
| Green digest buffer||2||2 | |||
|- | |||
| pTet mCh||5||5 | |||
|- | |||
| DpnI||1||0 | |||
|} | |||
<br><br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Thing''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | |||
|- | |||
| Digest buffer||4 | |||
|- | |||
| PCR rxn||25 | |||
|- | |||
|H2O||1 | |||
|- | |||
| DpnI||1 | |||
|} | |||
Incubated at 37°C for 55 minutes, sat on the bench for 3 minutes, heat inactivated at 80 for 5 minutes.<br> | |||
Cleaned using zymo DNA clean and concentration-5 kit (columns from gel extraction kit). Added 150ul of DNA binding buffer to DpnI reactions. | |||
<br> | |||
Gel extracted PCR reaction 1. Saw a smaller band above, think I got the correct band. | |||
<br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''PCR reaction''' | |||
| align="center" style="background:#f0f0f0;"|'''Description''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''260''' | |||
| align="center" style="background:#f0f0f0;"|'''280''' | |||
| align="center" style="background:#f0f0f0;"|'''260/280''' | |||
| align="center" style="background:#f0f0f0;"|'''ng/µL''' | |||
|- | |||
| 1||Rec bb||Gel extracted||0.02||0.01||2||20.289 | |||
|- | |||
| 2||pLux-GFP||DpnI digested, column purified||0.047||0.024||2||47.148 | |||
|- | |||
| 3||pRhl-GFP||DpnI digested, column purified||0.05||0.025||2.004||50.466 | |||
|- | |||
| 4||pEsa-GFP||DpnI digested, column purified||0.032||0.015||2.117||32.1 | |||
|- | |||
| 5||pLac-LuxR||DpnI digested, column purified||0.046||0.024||1.935||45.771 | |||
|- | |||
| 6||pLac-RhlR||DpnI digested, column purified||0.06||0.035||1.692||59.88 | |||
|- | |||
| 7||pLac-RhlR||DpnI digested, column purified||0.041||0.019||2.2||41.438 | |||
|- | |||
| 9||pLac-EsaR||DpnI digested, column purified||0.038||0.018||2.135||37.847 | |||
|- | |||
| | |||
|} | |||
Didn't have enough of the backbone so started another PCR. 1-4 are int receiver vector P112 P123, 5-6 are purified PCR product of receiver backbone P112 P123. | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | ||
|} | |} | ||
__NOTOC__ | __NOTOC__ |
Latest revision as of 23:56, 26 September 2017
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mm/dd/yyyyRan PCR products from yesterday on a gel. 5ul of reactions 2-9, all 30 of 1 so I can gel extract because there should be two bands and I only want one.
Incubated at 37°C for 55 minutes, sat on the bench for 3 minutes, heat inactivated at 80 for 5 minutes.
Didn't have enough of the backbone so started another PCR. 1-4 are int receiver vector P112 P123, 5-6 are purified PCR product of receiver backbone P112 P123. |