Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2013/10/28: Difference between revisions

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[[image:Pretty mRFP liq cultures.JPG|270px|Pretty mRFP]]
[[image:Pretty mRFP liq cultures.JPG|270px|Pretty mRFP]]


<br>
<br><br>
'''Miniprep'''<br> 33ng/ul. <br>
'''Restriction digest'''<br>
cut 15ul of 33ng/ul, so about 0.5ug of plasmid. Cut with E/X in a 20ul rxn for 25 minutes at 37°C<br>  


Gel [[image:13.10.28_J04450.png‎|J04450 E/X]]
Gel of cut J04450 [[image:13.10.28_J04450.png‎|J04450 E/X]]
 
<br>Cut out and put the gel slice at -20°C<br><br>
 
PCR ''need to edit this to include the next lane'' [[image:13.10.28_PCR_cropped.png‎|PCR]]


PCR ''need to edit this to include the next lane. lane 1 is P087/P088 and lane 2 is P089/P090, both I13522'' [[image:13.10.28_PCR_cropped.png‎|PCR]]
<br><br>
Ran another PCR reaction to text which primer wasn't working for the sender backbone. Used the two sender backbone primers, P089 and P090 with the matching F or R primer from the pLux vectors primers which were shown to work. Rxn 3 is positive control.
1
:15ul reaction
:P087/P090
2
:15ul reaction
:P089/P088
3
:10ul
:P087/P088


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Revision as of 15:23, 15 November 2013

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mm/dd/yyyy

Miniprepped J04450 that I started growing on Saturday. They were in the incubator for a long time and were super red.

Pretty mRFP



Miniprep
33ng/ul.
Restriction digest
cut 15ul of 33ng/ul, so about 0.5ug of plasmid. Cut with E/X in a 20ul rxn for 25 minutes at 37°C

Gel of cut J04450 J04450 E/X
Cut out and put the gel slice at -20°C

PCR need to edit this to include the next lane. lane 1 is P087/P088 and lane 2 is P089/P090, both I13522 PCR

Ran another PCR reaction to text which primer wasn't working for the sender backbone. Used the two sender backbone primers, P089 and P090 with the matching F or R primer from the pLux vectors primers which were shown to work. Rxn 3 is positive control. 1

15ul reaction
P087/P090

2

15ul reaction
P089/P088

3

10ul
P087/P088


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