Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2013/10/09: Difference between revisions
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Rene M Davis (talk | contribs) (Autocreate 2013/10/09 Entry for Haynes_Lab:Notebook/Characterizing_AHL_quorum_sensing_homologs) |
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==mm/dd/yyyy== | ==mm/dd/yyyy== | ||
'''Cloning''' | |||
Cut 10ul J06702 (131) with E/X in 20ul reaction<br> | |||
Cut Gblock pLac-RBS with E/S<br> | |||
Used the FD buffer that doesn't have dye<br> | |||
Cut for 15 min at 37deg<br> | |||
purified both with Zymo OligoClean & Concentrator, eluted in 15ul water<br> | |||
''realized later that this won't work for the vector because uncut vector will be purified with cut vector. led to very high background''<br> | |||
{| {{table}} | |||
|- | |||
|Part||Enzymes||Concentration||Length | |||
|- | |||
|J06702|| E/X||75.2 ng/ul||3024bp | |||
|- | |||
|Gblock||E/S||2.6ng/ul||~80bp | |||
|- | |||
|} | |||
Insert:backbone ratios 1:1, 2:1, 3:1 | |||
{| {{table}} | |||
|- | |||
| ||1:1||2:1||3:1||Bb ctrl | |||
|- | |||
|Insert||0.5||1||1.5||0 | |||
|- | |||
|Vector||0.66||0.66||0.66||0.66 | |||
|- | |||
|2x ligation buffer||5||5||5||5 | |||
|- | |||
|Ligase||1||1||1||1 | |||
|- | |||
|Water||3||2.5||2||3.5 | |||
|- | |||
}} | |||
Incubate at RT for 35min<br> | |||
Add 40ul DH5αT cells to entire rxn<br> | |||
Incubate on ice for 30 mins<br> | |||
heat shock at 42°C for 30s<br> | |||
ice 2min<br><br> | |||
'''GG PCR''' | |||
Ran PCR from 10/8 on gel<br> | |||
Reporter (pLux) primers worked, no background<br> | |||
Sender did not work. Retried with I13522-2 | |||
Revision as of 15:57, 15 October 2013
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mm/dd/yyyyCloning
Cut 10ul J06702 (131) with E/X in 20ul reaction
Insert:backbone ratios 1:1, 2:1, 3:1 }} Incubate at RT for 35minAdd 40ul DH5αT cells to entire rxn Incubate on ice for 30 mins heat shock at 42°C for 30s ice 2min GG PCR Ran PCR from 10/8 on gel Reporter (pLux) primers worked, no background Sender did not work. Retried with I13522-2
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