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Team:Newcastle/Slide Preparation
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| - | + | = Slide Preparation = | |
| - | #Grow overnight culture in | + | #Grow overnight culture in 5 ml of LB broth, with appropriate antibiotics if required, at 37°C. |
| - | #Dilute the overnight culture to a ratio of 1:100 | + | #Dilute the overnight culture to a ratio of 1:100 (overnight culture:LB with appropriate antibiotic ). Incubate at 37°C for 1 hour. |
| - | #Set up the following broths with different concentrations of IPTG in | + | #Set up the following broths with different concentrations of IPTG in 5 ml of LB broth: |
#* 2mM IPTG | #* 2mM IPTG | ||
| + | #* 1mM 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 containing the different concentrations of IPTG with the appropriate cultures and incubate at 37°C for two hours. |
| - | #Prepare the slides by pippeting 500 | + | #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. | #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. | ||
#Transfer 200 µl of each sample to microfuge tubes and label accordingly. Add 0.5 µl of membrane dye to each sample. | #Transfer 200 µl of each sample to microfuge tubes and label accordingly. Add 0.5 µl of membrane dye to each sample. | ||
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#The slide is now ready for microscopy. | #The slide is now ready for microscopy. | ||
| - | + | '''Go back to our [[Team:Newcastle/Protocol list|Protocol List]]''' | |
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{{Team:Newcastle/footer}} | {{Team:Newcastle/footer}} | ||
Latest revision as of 17:02, 26 October 2010
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Slide Preparation
- Grow overnight culture in 5 ml of LB broth, with appropriate antibiotics if required, at 37°C.
- Dilute the overnight culture to a ratio of 1:100 (overnight culture:LB with appropriate antibiotic ). Incubate at 37°C for 1 hour.
- Set up the following broths with different concentrations of IPTG in 5 ml of LB broth:
- 2mM IPTG
- 1mM IPTG
- 0.2mM IPTG
- 0.02mM IPTG
- Broth only
- Innoculate the broth containing the different concentrations of IPTG 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.
- Transfer 200 µl of each sample to microfuge tubes and label accordingly. Add 0.5 µl of membrane dye to each sample.
- Place 0.5 µl of each sample in each well of the slide and label accordingly. Place a coverslip on top of the slide.
- The slide is now ready for microscopy.
Go back to our Protocol List
   
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