From 2013.igem.org
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| - | "date" : "2013-8-30", | + | "date" : "2013-08-15", |
| - | "author" : "fabio", | + | "author" : "emil", |
| - | "title" : " <html> new experiments on blue light induction: great!!! </html> ", | + | "title" : "Cheking integration(2)", |
| - | "content" : " <html> In these days I tried so many times to induce transformed cells in order to have great, clean, results, and finally I found the key: I developed a standard procedure that allowed me to see difference between samples. I transformed NEB10b cells with the entire construct (first I tested different strains of E. coli in order to see which strain had the best behavior and noticed that NEB10b cells work better than any other). Then I made some inocula O/N using LB broth (I also tried to compere LB broth to M9 minimal medium, but I didn’t see any difference in the outcome) and diluted the next morning in 20 ml of liquid broth (1:50) waiting until they reached OD = 0.7. The thing is, cultures grew really slowly, in fact they reached the required concentration after 6-8 hours. Then I split them into 5ml samples that I exposed to different condition. The first time that I performed this experiment I tested different light sources in order to establish the most powerful inducing condition: I used blue LEDs, a blue bulb light and normal white light. I finally picked out the LED and the normal light for further experiments. Now let’s go back to the different samples!! I used glass tubes for overnight induction, heating the samples at 37 degrees with stirring. Previously I tested different materials (glass and plastic) tubes and different temperatures conditions: the best way to grow my bacteria was definitely in glass at 37 degrees. I put one sample in the dark wrapping it up with an aluminum foil and placed under a box to be sure that no light could pass. The second sample was illuminated using a blue LED. Instead the third one was exposed to normal white light because I previously saw that even white light induces the circuit. Experiments lasted all night long and everytime I saw the results in the morning. Finally I got my results right in front of my eyes: the dark control in almost every experiments didn’t show any sign of amilCP production. At the other hand the other two samples were blue. To obtain quantitative measurements I used the spectrometer. First I diluted the pellets in 2 ml of PBS and then sonicated the samples for 10 seconds. Fluorescence spectra reveals what we already got from visual results. Great!!</html> ", | + | "content" : "<html> The inocula of yesterday were positive as you can see from the image:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Test image |<html><img src='https://static.igem.org/mediawiki/2013/6/65/UqMLuy3LMCXLFRZ-ZQqyoljqsZZMxXTmzBmOuMfWye4.jpeg'/>}}<html> Unfortunately the GFP was incorrect so I decided to repeat the experiment with the medium previously conserved at -20 C°.I tested two plates from THomas and Viola: PXyl+GFP and PXyl+EFE.</html>", |
| - | "tags" : "blue_light" | + | "tags" : "B.subtilis-PXy+GFPl" |
| | } | | } |
Latest revision as of 09:14, 3 October 2013
{
"date" : "2013-08-15",
"author" : "emil",
"title" : "Cheking integration(2)",
"content" : " The inocula of yesterday were positive as you can see from the image:{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Test image |
}} Unfortunately the GFP was incorrect so I decided to repeat the experiment with the medium previously conserved at -20 C°.I tested two plates from THomas and Viola: PXyl+GFP and PXyl+EFE.",
"tags" : "B.subtilis-PXy+GFPl"
}
"
