Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/02/13: Difference between revisions

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:Incubate the two tubes at 37°C for 10 minutes
:Mix, spin, and incubate the two tubes at 37°C for 10 minutes
:Close all caps and return everything else to your box in the freezer
:Close all caps and return everything else to your box in the freezer
<br>
<br>
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:Bring the rApid Alkaline Phosphatase from you box to the bench in the ice box
:Bring the rApid Alkaline Phosphatase from you box to the bench in the ice box
:Add 2ul of phosphatase to each digestion reaction.  
:Add 2ul of phosphatase to each digestion reaction.  
:Incubate at 37°C for 15-60 mins  
:Mix, spin, and incubate at 37°C for 15-60 mins  
::''if you're short on time, you can do 15, if you have more time, I recommend a little longer for more complete dephosphorylation''
::''if you're short on time, you can do 15, if you have more time, I recommend a little longer for more complete dephosphorylation''
:Close all caps and return everything else to your box in the freezer
:Close all caps and return everything else to your box in the freezer
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'''Step 3: Purify cut backbone'''
'''Step 3: Purify cut backbone'''
:Combine the two digested and dephosphorylated samples into one column and elute in 20ul like you did today with the Sigma PCR purification kit  
:Combine the two digested and dephosphorylated samples into one column and elute in 20ul like you did today with the Sigma PCR purification kit  
:Measure the concentration and put in your box
:Measure the concentration and put in your box in the freezer


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Revision as of 16:22, 12 February 2015

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02/12/2015

Step 1: Cut backbone with EcoRI and PstI

Make sure the small heat block is set to 37°C with the smallest tube size block inside
Ryan aliquoted the backbone into two PCR tubes with 1ug of backbone in each tube and put them in your box
Get from your box
The two backbone tubes
Fast digest buffer
PstI
EcoRI
be sure to put the enzymes in the cold box when you bring them out of the freezer
Add the following directly to the tube with the backbone:
Thing vol in ul
1ug sample 6
H2O 10
10x buffer 2
PstI 1
EcoRI 1
Total 20
Mix, spin, and incubate the two tubes at 37°C for 10 minutes
Close all caps and return everything else to your box in the freezer


Step 2: Dephosphorylate cut ends

Bring the rApid Alkaline Phosphatase from you box to the bench in the ice box
Add 2ul of phosphatase to each digestion reaction.
Mix, spin, and incubate at 37°C for 15-60 mins
if you're short on time, you can do 15, if you have more time, I recommend a little longer for more complete dephosphorylation
Close all caps and return everything else to your box in the freezer


Step 3: Purify cut backbone

Combine the two digested and dephosphorylated samples into one column and elute in 20ul like you did today with the Sigma PCR purification kit
Measure the concentration and put in your box in the freezer


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