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

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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
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


These are the results obtained after eluting the dephosphorylated and digested backbone twice. The first elution has a much higher concentration than the second elution, which is what we expected.

Sample Read# 260 280 260/280 ng/µL
1 (first elution) 0.063 0.032 1.934 62.779
2 (second elution) 0.006 0.002 2.81 6.212