After performing the transformation experiment, I have collected the seeds. I now want to select the transformants from the seeds collected. So, I tried to sterilize the seeds with NaOCl and SDS. But, when I mixed 800µL of 5% NaOCl with 200µL of 2% SDS (to make 4% NaOCl +1% SDS), the solution turned turbid (cottage cheese like appearance). Is it usual, or is something amiss? Has anybody experienced something similar?
Perhaps you should try a (few) drop(s) of Tween in stead of SDS as a detergent!
Add 1 ml of 0.1% Triton X-100 (or 5-7% Bleach + 0.1% Triton X-100) 3min. Invert the tubes 3-4 times to expose all seeds. Do not leave seeds in bleach for longer than 3 minutes as this will cause seed coat breakdown.
You could try hydrogen peroxide as a sterilant instead of NaOCL and as Tobias suggests use Tween 20 or Tween 80 as your adjuvant to the solution. Tween is usually preferred because it is a nonionic detergent. Usual concentration of H2O2 is 3-5% for sterilization. Depends on the sensitivity of your seed.
I usually followed this protocol.
Nature Protocols 1, 641 - 646 (2006)
"Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method"
They recommended
1. Wash with 50X volume of 70% ethanol for 1 min.
2. Wash with 50X volume of 50% bleach/50% water/0.05% Tween for 10 min and vortexing the suspension vigorously every 2 min.
I use the protocol described above by Mohammad Ali and works perfectly! I leave the seeds for 15 min and then wash three times with sterile water. Good luck!
As an alternative to the use of bleach (and to keep the seeds sterile AND dry) you could also try sterilization using dichloroisocyanuric acid. Protocol here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.0040143
I do a 1st wash with etanol 96%, for 1 min to remove wax and them the Mohammad Ali protocol.
Simple surface sterilization of Arabidopsis seeds using 0.1% SDS for 5-6 minutes, 70% ethanol treatment for 2 minutes followed by five times wash with sterile distill H2O is effective in Arabidopsis and have worked all the times in our case. Best wishes :-)
The protocol that we use to sterilize Arabidopsis seeds is based on Cl2 gas formation. Easy, fast and efficient!
Work in a glass jar in the fume hood:
- Fill the bottom of Eppendorf tubes with your seeds, place them with open lid in a holder in the jar.
- Add 3 ml HCl to 100 ml Javel in a flask placed into the jar. Be carefull: the reaction starts immediately, so already hold the lid of the jar in the other hand to close it as quickly as possible.
- Incubate for 4 hours
- Then open the jar, take out yours tubes, close them quickly and sow them under the laminar flow.
Succes!
For A.th. seed sterilization, we use a 1-2 min wash with 70% ethanol, than the Mohammad Ali protocol, it works well!
We use the following protocol for Arabidopsis:
Seed aliquots are placed in clean 1.5 mL microcentrifuge tubes. Seeds are rinsed with 500 µl of 100% ethanol for 2 min. Mix well!
Then let seed settle, or spin for 10 sec. Remove ethanol and rinse seeds with 500 µl of bleach solution (50% bleach, 50%water, 0.05%tween) for 2 min. Mix well! Avoid letting seed sit in bleach for longer time, you might kill them.
The bleach solution is removed and seeds are washed with 1mL of sterile water.
The washing step is repeated 2-3 times.
Finally the seeds are suspended in sterile 0.01 % Agar and plated on MS plates (+antibiotics if needed).
Good luck!
I used for decontamination of thymus spp. seeds this protocol and I obtained 100% decontaminated material;
The seeds were surface sterilized by soaking in 70% ethanol for 5 min and disinfected by a 5-min treatment with 10% hydrogen peroxide and a 15-min treatment with 20% commercial bleach (at 2% active chlorine, Domestos, Unilever, London, UK), with consecutive rinses in sterile distiled water after each step. (this procedure was performed in eppendorf tubes because thymus seeds are so small)
70% ethanol for 5 min , then 100% ethanol 2 min, dry on the filter paper.
I performed a lot of experiments with the A. thaliana seeds where was necessary have sterile materials. I used the below protocol with success:
Sterilization of Arabidopsis seeds
1. Made 10 ml of 80% EtoH in 15ml plastic tube and added 500mg Calcium hypochloride (i.e. 5%) and 2µL of Triton X-100 (i.e. 0.02%). Shake the tube.
2. Take about 100 seeds and add 1ml of above solution.
3. Shake well at 25-280 C for 10-12 min.
4. Pipette out the liquid carefully. Don’t waste the seeds.
5. Wash with 80% ethanol two times.
6. Wash with 100% ethanol.
7. Dry the seeds if need to be kept for long time. Otherwise wash several times with sterile water and add appox. 1 ml water and keep in fridge if need to use in next 2-3 days.
8. Next day spread on agar plate.
Good luck
Ps: the step 7 include the vernalization (for synchronization of the germination) procedure
Sorry in the step 3 there is a typos error. Please rectify with this:
3. Shake well at 25-28° C for 10-12 min.
The standard protocol that we use for sterilization of Arabidopsis seeds is:
In a microfuge tube aliquot seeds
Ethanol (95 - 100%) - 1 min
Rinse with sterile water
Sterilize with 1ml of 20% Bleach with 0.05% Tween 20 for 15 min with occasional mixing
Rinse with sterile water (4x, 5 min each) - its not only the number of washes, but also the duration of the washes that are important to get rid of the bleach.
This above protocol works very well for us.
Best wishes
As I think many have said already, Ethanol is increadibly convenient and works great! (We use 95% + 0.1% tween for 3 min, followed by 3-5 washes with 75% Ethanol).
Alternative, bleach also works well. 3 min 70% Ethanol followed by 15 min 4% bleach with tween and then 4-6 washes with water. This takes longer and we haven't found it much better than Ethanol sterilization for routine work, but its certainly a bit harder, so might be useful in ocasions.
Hope this helps!
I have tried sterilizing Arabidopsis seeds in multiple ways. The absolute easiest and fastest way I have found comes from a postdoc with whom I used to work--Mee Yeon Park--and I never get contamination. All work is done in a laminar flow hood.
1. I put the seeds to be sterilized in a 1.5 ml tube and add about 1 ml of 70% ethanol. I invert the tubes to mix until all of the seeds are coated and there are no clumps of seeds.
2. All the seeds to sit in the 70% ethanol for 10-15 minutes, inverting periodically.
3. Remove the 70% ethanol, add about 500 ul-1 ml of 100% ethanol, invert to mix, and immediately pipet the seeds and ethanol onto a piece of sterile filter paper that has been creased in half.
4. Allow the seeds to dry. This does not take long because 100% ethanol evaporates quickly.
5. Once dry, you can pour the seeds into a new sterile 1.5 ml tube, and plant as desired.
Hope this helps.
Try a 2.5 - 5.0 % solution of domestic bleach, if the bleach has not detergent in it add roughly 0.1 % Tween-20 (one drop in 100 ml) to wet the seeds, especialy if trying to sterilise them straight from the selique. mix for 5 minutes then wash 3X in sterile water, and plate out. Remeber bleach will break dormancy and will kell the seeds if left fot too long. Also you cannot conpletely surface sterilise seeds which out damaging them so beware if plating seeds on to media containing sugars, unless they are germination mutants sugars are not needed. Also avaid ethanol if possible as this will also break dormancy and kill the seeds if used excerssively. DO NOT USE SDS. It might be a universal detergent for molecular biologists but not for seed biologists.
Many seeds actually require harsh treatments to germinate. It will always depends on the species you are using. I also use ethanol and works great. Ethanol and Triton, in my case. But very similar to the Ethanol-Tween treatments described above. Since you are talking Arabidopsis. This should work fine. Other seeds it may not.
For those seeds sterilization normally try this simple protocol:
1) Put the seeds in water with 2% domestical bleach, during 30 to 40 minutes, keep them shaking manually or with magnetic shaker. (This first step is just necessary if you took the seeds right from the ground)
2) Wash the seeds with destilled water to eliminate the bleach (if you did the step above). From this step work with the laminar flow chamber.
3) With a sterilized glass, drown the seed in a solution of ethanol at 70% for 30 seconds only.
4) Wash the seeds 3 times with destilled water to eliminate the ethanol.
5) Transfer the seeds to some tubes and drown them in a solution of Sodium hypochlorite at 1% (dechlorinated) during 10 minutes.
6) Wash the seeds five times with destilled water to eliminate the sodium hypochlorite and decant helping with a spatula.
Then you just have to do the seed sowing and incubation, or what ever you have to do.
I know it's late and you aready had success, but hopefully works for someone else!
sterilized by incubating them in 1 ml of 70% ethanol for 10 minutes, followed by 1 ml 6% bleach in 0.1% Tween20 for 15 minutes. Bleach hinders seed growth on plates and therefore the seeds must be thoroughly washed five times with autoclaved distilled water.
Dear Banerjee,
0.6 or 0.4 % of sodium hypochlorite is used for sterlization for time 10 minutes. SDS or triton X is added as sufectant. you have to add a tiny drop for 50ml of 0.4 or 0.6 % of sod hyp chlorite solution. after 10 min sterlization u have to wash with autoclaved water 3 times.
We sterilized arabidopsis seeds for the following obtaining tissue and suspension cultures. Take necessary amount of seeds and place them in Eppendorf tube. Add 70% ethanol for 30 sec and remove it with automatic pipette. Then add 900 mcl of sterile water (previously contained 0.01% Tween 20) and 100 mcl 10% NaOCl. Shake vigorously 15 min. Then wash seeds with sterile water 3 times for 5 min. Remove last portion of water and put in tube sterile 0.1-0.2% agar, mix with seeds and disperse them by microdrops on nutrient agar medium. Each microdrop may contain 1-2 seeds.
6% bleach in EtOH for 5 min (shaking)
remove liquid
rapidly shake for few seconds in 96% EtOH in dH2O
remove liquid
rapidly shake for few seconds in 100% EtOH (absolute) in dH2O
hold your tube sideways so seeds are spread
remove liquid
leave to dry in laminar flow (facing the air stream)
works fine!
The turbidity and "cottage cheese" like precipitates are most likely the SDS going out of solution.
There are a lot of different methods of "Sterilization of Arabidopsis Seeds" , and there was another recent discussion in this forum a few months back. Most (if not all) of them works fine. Ethanol, bleach solution etc are commonly used. Please see the previous discussion for choosing one.
Place seeds (approx. 20-50 mg) into Eppendorf flask (1.5 ml) + 9 parts of sterile water and 1 part of 10% NaOCl + 0.1% Tween 20. Shake 10-15 min. Wash 3 times for 5 min with sterile water. The seeds will be sterile and may be sow. In order to sow I suspend the seeds in 0.15-0.2% agar solution, take portions 10 mcl and put them on the soil surface. It is possible to take 1-2 seeds in each portion.
In may case, 0.02% sodium hypochlorite worked well for Arabidopsis seeds.
1. 70% ethanol for 5 mins
2. absolute ethanol for 1min
3. Dry it under laminar hood.
Method using silica membrane columns (no pipetting)
Find the protocol in enclosed pdf, here is the text:
• Get (used) silica membrane columns used for RNA/DNA purification (Qiagen, MN, …) and their collection tube
• Store columns and collection tubes in 70% EtOH, take out the number you need and dry them
• Add the seeds into the column (never fill the column more than half)
• Add 700 ul of 70% EtOH, close quickly the lid of the column, otherwise the EtOH goes directly through the column
NB: If some columns let the ethanol go through even if lid is closed properly, add again ethanol and discard the flow through from the collection tube. Usually the ethanol should stay in the column the second time
• Keep shaking the tubes by hand for 7 min (alternatively, use Qiagen TissueLyser machine set at 8 Hz)
• Spin down the columns 1 min full speed at RT
• Discard the flow through from the collection tube and replace the column
• Add 700 ul of 100% EtOH and close quickly the lid of the column
• Keep shaking by hand the tubes for 5 min (alternatively, use Qiagen TissueLyser machine set at 8 Hz for shaking)
• Spin down the columns 1 min full speed at RT
• Discard the flow through and repeat the centrifugation to get rid of the residual EtOH (seeds will be completely dry)
Notes:
• Sow the seeds on the same day
• If long storage is needed, transfer the seeds in sterile microcentrifuge tubes and close the lid properly
• You have sterile seeds now, only open the column/tube under the laminar flow hood
It is not necessary to use so high NaOCl concentration and 2% SDS. 0.5-1% NaOCl and one drop of Tween-20 for 100 ml is sufficient for Arabidopsis seeds
sodium hypochlorite (3%) for 10 minutes, to remove the hypochlorite excess, wash with sterile distilled water.
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