Difference between revisions of "Project:Plant species testing"

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We used two methods of extraction. Method 1:
 
We used two methods of extraction. Method 1:
  
* Blend peas with salt and detergent
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* Blend peas with salt and detergent according to [http://learn.genetics.utah.edu/content/labs/extraction/howto/ this] protocol. Sieve.
* Incubate for 15 mins at 60 C (optional step not taken)
+
* Incubate for 15 mins at 60°C (optional step that we didn't take)
* Filter and centrifuge - recover supernatant
+
* Centrifuge - recover supernatant
* Add proteinease K (0.8 ul - incubate for 1 hr at 50C then 94 C for 10 mins (inactivation of proteinase K)
+
* Add proteinease K (100 ul of 100mg / ml to 300 ul pea template) - incubate for 1 hr at 50C (action of proteinase k) then 94 C for 10 mins (inactivation of proteinase K)
* Precipitate with rubbing alcohol and meat tenderiser as an enzyme
+
* Precipitation to follow
* Centrifuge and remove supernatant to leave pellet
 
  
Method 2:
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Method 2 (From [http://www.springerlink.com/content/n6293v8808n5g836/ this] paper - we also have full text available):
  
* 'Homogenize' with a pestle in 5% chelex solution for 1 min   
+
* 'Homogenize' (mush) approx 40 mg pea with a pestle in 200ug of 5% chelex solution for 1 min   
* Vortex for 10s then boil for 5 mins, then vortex again for 10s
+
* Vortex for 10s then boil for 5 mins, then vortex again for 10s. (Hot plate lost heat in the middle so we boiled for 8 mins)
 
* Centrifuge for 1 min - recover supernatant and use as template
 
* Centrifuge for 1 min - recover supernatant and use as template
 +
 +
PCR:
 +
 +
* Prepare sample with 12.5 ul of Taq ready mix, 2 ul of each primer, 3 ul of template, and 5.5 ul of distilled water.
 +
* Denature at 96°C for 5 mins
 +
* 40 cycles of D = 96°C for one minute, A = 58°C for 30 seconds, E = 72°C for one minute.
 +
* Put paraffin on top after 5 mins (should have been before start)

Revision as of 22:30, 17 July 2012

We are using primers for plants to test and refine our DNA extraction / PCR technique. First project is to test for peas. Inspiration in this paper.


First extraction and PCR - 17/7/12

We used two methods of extraction. Method 1:

  • Blend peas with salt and detergent according to this protocol. Sieve.
  • Incubate for 15 mins at 60°C (optional step that we didn't take)
  • Centrifuge - recover supernatant
  • Add proteinease K (100 ul of 100mg / ml to 300 ul pea template) - incubate for 1 hr at 50C (action of proteinase k) then 94 C for 10 mins (inactivation of proteinase K)
  • Precipitation to follow

Method 2 (From this paper - we also have full text available):

  • 'Homogenize' (mush) approx 40 mg pea with a pestle in 200ug of 5% chelex solution for 1 min
  • Vortex for 10s then boil for 5 mins, then vortex again for 10s. (Hot plate lost heat in the middle so we boiled for 8 mins)
  • Centrifuge for 1 min - recover supernatant and use as template

PCR:

  • Prepare sample with 12.5 ul of Taq ready mix, 2 ul of each primer, 3 ul of template, and 5.5 ul of distilled water.
  • Denature at 96°C for 5 mins
  • 40 cycles of D = 96°C for one minute, A = 58°C for 30 seconds, E = 72°C for one minute.
  • Put paraffin on top after 5 mins (should have been before start)