Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2013/05/30

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(Entry title)
(Entry title)
Line 34: Line 34:
Supernatant transfer:
Supernatant transfer:
 +
Spun down 4ml of culture in 2-2ml tubes for each sender. Poured supernatant into round bottom tubes. Dropped some supernatant onto pH strip. Added 1ml of receiver cells to a 2ml centrifuge tube. Added 20ul of Amp and shook. Added 100ul of Receivers + (~4ul) Amp to each 4ml volume of Sender cells in round bottom tubes. Added 1ml of LB+Amp to add nutrients and lower the pH a little. Same Sender-Receiver pairs as above. Started shaking ~10:30.
 +
<br>
Determine pH using test strips:
Determine pH using test strips:
{| {{table}}
{| {{table}}
Line 39: Line 41:
|Sender||pH
|Sender||pH
|-
|-
-
|LuxI||
+
|Plain LB||6-7
|-
|-
-
|LasI||
+
|LuxI||7-8
|-
|-
-
|RhlI||
+
|LasI||7-8
|-
|-
-
|TraI||
+
|RhlI||7-8
|-
|-
-
|TraR ctrl||
+
|TraI||7-8
 +
|-
 +
|TraR ctrl||7-8
|-
|-
|}
|}
 +
<br>

Revision as of 13:31, 30 May 2013

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Entry title

9AM

BL21 sender cultures grew in 6mls and 2mls. RhlR-GFP still didn't grow.
Shook LuxR, TraI, and TraR that had been in the fridge for a few minutes.

Co-cultures: Add 100ul of Sender, 100ul of Receiver to 3ml LB+Amp (except for TraI culture, which will not have antibiotic). Started shaking at 10AM.

SenderReceiver
LuxILuxR
LasILuxR
RhlILuxR
TraILuxR
TraR ctrlLuxR



Supernatant transfer: Spun down 4ml of culture in 2-2ml tubes for each sender. Poured supernatant into round bottom tubes. Dropped some supernatant onto pH strip. Added 1ml of receiver cells to a 2ml centrifuge tube. Added 20ul of Amp and shook. Added 100ul of Receivers + (~4ul) Amp to each 4ml volume of Sender cells in round bottom tubes. Added 1ml of LB+Amp to add nutrients and lower the pH a little. Same Sender-Receiver pairs as above. Started shaking ~10:30.
Determine pH using test strips:

SenderpH
Plain LB6-7
LuxI7-8
LasI7-8
RhlI7-8
TraI7-8
TraR ctrl7-8





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