Team:Purdue/Seed sterilization

From 2010.igem.org

(Difference between revisions)
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Seed Sterilization
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==Solutions==
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#Sterile distilled water
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#70% ethanol
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#50% bleach/1 drop Tween 20: prepare 40 ml in a Falcon tube, use bleach that is less than two months old
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[[Solutions]]
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==Procedure==
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1. Sterile distilled water
+
#transfer a small scoop of seed to an eppendorf tube; soak seed for 30 minutes in distelled water
 +
#spin down seed briefly in microfuge
 +
#decant water, add 500 uL 70% EtOH, vortex, spin
 +
#decant EtOH, rinse with sterile water, spin and decant
 +
#add 0.8 mL 50% bleach, vortex for 8-10 min, spin and decant
 +
#add 0.8 mL sterile water, vortex, spin, decant
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#repeat Step 6 six times
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#leave seed in 0.5 mL of 0.12% sterile agar; allow to stratify at 4 degrees for at least 2 days
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2. 70% ethanol
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==Notes==
-
3. 50% bleach/1 drop Tween 20: prepare 40 ml in a Falcon tube, use bleach that is less than two months old
+
#keep the ethanol step as brief as possible-prolonged exposure to EtOH decreases germination
-
 
+
#multiple water washes are to remove all traces of bleach which also affects germination
-
[[Procedure]]
+
#do the procedure in a hood in the tissue culture room
-
 
+
#store washed seed for stratification in refrigerator vegetable drawer in the prep room
-
1. transfer a small scoop of seed to an eppendorf tube; soak seed for 30 minutes in distelled water
+
-
 
+
-
2. spin down seed briefly in microfuge
+
-
 
+
-
3. decant water, add 500 uL 70% EtOH, vortex, spin
+
-
 
+
-
4. decant EtOH, rinse with sterile water, spin and decant
+
-
 
+
-
5. add 0.8 mL 50% bleach, vortex for 8-10 min, spin and decant
+
-
 
+
-
6. add 0.8 mL sterile water, vortex, spin, decant
+
-
 
+
-
7. repeat Step 6 six times
+
-
 
+
-
8. leave seed in 0.5 mL of 0.12% sterile agar; allow to stratify at 4 degrees for at least 2 days
+
-
 
+
-
[[Notes:]]
+
-
 
+
-
1. keep the ethanol step as brief as possible-prolonged exposure to EtOH decreases germination
+
-
 
+
-
2. multiple water washes are to remove all traces of bleach which also affects germination
+
-
 
+
-
3. do the procedure in a hood in the tissue culture room
+
-
 
+
-
4. store washed seed for stratification in refrigerator vegetable drawer in the prep room
+

Revision as of 14:29, 8 July 2010

Solutions

  1. Sterile distilled water
  2. 70% ethanol
  3. 50% bleach/1 drop Tween 20: prepare 40 ml in a Falcon tube, use bleach that is less than two months old

Procedure

  1. transfer a small scoop of seed to an eppendorf tube; soak seed for 30 minutes in distelled water
  2. spin down seed briefly in microfuge
  3. decant water, add 500 uL 70% EtOH, vortex, spin
  4. decant EtOH, rinse with sterile water, spin and decant
  5. add 0.8 mL 50% bleach, vortex for 8-10 min, spin and decant
  6. add 0.8 mL sterile water, vortex, spin, decant
  7. repeat Step 6 six times
  8. leave seed in 0.5 mL of 0.12% sterile agar; allow to stratify at 4 degrees for at least 2 days

Notes

  1. keep the ethanol step as brief as possible-prolonged exposure to EtOH decreases germination
  2. multiple water washes are to remove all traces of bleach which also affects germination
  3. do the procedure in a hood in the tissue culture room
  4. store washed seed for stratification in refrigerator vegetable drawer in the prep room
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