Team:Newcastle/Slide Preparation
From 2010.igem.org
(Difference between revisions)
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#Dilute the overnight culture to a ratio of 1:100 and incubate at 37C for 1 hour | #Dilute the overnight culture to a ratio of 1:100 and incubate at 37C for 1 hour | ||
#Set up the following broths with different concentrations of IPTG in 5ml of LB broth | #Set up the following broths with different concentrations of IPTG in 5ml of LB broth | ||
- | + | #* 2mM IPTG | |
- | * 2mM IPTG | + | #* 0.2mM IPTG |
- | * 0.2mM IPTG | + | #* 0.02mM IPTG |
- | * 0.02mM IPTG | + | #* Broth only |
- | * Broth only | + | |
- | + | ||
#Innoculate the broth with the appropriate cultures and incubate at 37°C for two hours. | #Innoculate the broth with the appropriate cultures and incubate at 37°C for two hours. | ||
#Prepare the slides by pippeting 500 µL of 1.2% agarose. Gently place the coverslip over the slide. | #Prepare the slides by pippeting 500 µL of 1.2% agarose. Gently place the coverslip over the slide. | ||
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#Place 0.5 µl of each sample in each well of the slide and label accordingly. Place a coverslip on top of the slide. | #Place 0.5 µl of each sample in each well of the slide and label accordingly. Place a coverslip on top of the slide. | ||
#Transfer 200 µl of each sample to microfuge tubes. | #Transfer 200 µl of each sample to microfuge tubes. | ||
- | #Before loading the sample add 1 µl of membrane dye | + | #Before loading the sample add 1 µl of membrane dye to each sample. |
+ | |||
- | |||
Revision as of 08:39, 23 September 2010
|
- Grow overnight culture in 5ml of LB broth at 37C
- Dilute the overnight culture to a ratio of 1:100 and incubate at 37C for 1 hour
- Set up the following broths with different concentrations of IPTG in 5ml of LB broth
- 2mM IPTG
- 0.2mM IPTG
- 0.02mM IPTG
- Broth only
- Innoculate the broth with the appropriate cultures and incubate at 37°C for two hours.
- Prepare the slides by pippeting 500 µL of 1.2% agarose. Gently place the coverslip over the slide.
- Wait for about 5 minutes for the agarose to harden. Remove the coverslip by gently sliding the coverslip horizontally from the slide. This is to ensure that agarose layer remains undisturbed.
- Place 0.5 µl of each sample in each well of the slide and label accordingly. Place a coverslip on top of the slide.
- Transfer 200 µl of each sample to microfuge tubes.
- Before loading the sample add 1 µl of membrane dye to each sample.