2024年6月1日发(作者：)

- Plastic to cover bench + absorbant paper

- 塑料纸和吸水纸（大滤纸）铺在实验台上

- Plastic beakers + plastic bag: 2x + NaOH

- 塑料烧杯、塑料袋两个 和NaOH溶液

- 200µl pipette + parafilm +filter tips, blue tips, ster. Eppendorfs

- 200µl 移液器+保鲜膜+带过滤嘴枪头，蓝色枪头+无菌离心管

- Shaker + parafilm

- 振荡器（封口膜包住）

- Strips of parafilm

- 一卷封口膜·

- 15 ml falcon, pasteur pipette巴斯德吸管

- Mark door and fume hood 通风橱

- Ster. H

2

O, 100 ml 100mM phosphate buffer (KHPO

4

), 0,05% triton X, 5M NaOH (5

liter)

- 无菌水，100ml 100mM KHPO4，0.05% triton X, 5M NaOH

- 转子，磁力搅拌器，100ml锥形瓶

Potassium phosphate buffer (pH 7.5)

- 100ml 1M K

2

HPO

4

/(HK2O4P.3H2O) and 100ml 1M KH

2

PO

4

174.18g/mol (228.22g/mol) 136.1g/mol

取70ml 1M K

2

HPO

4

于烧杯中，加入20ml 1M KH

2

PO

4

，混匀，用剩

余的1M KH

2

PO

4

调节pH=7.5，稀释10倍，即为100mM 磷酸缓冲液

100mM Na

2

S

2

O

3

（硫代硫酸钠）

Na

2

S

2

O

3

.5H

2

O

100ml

248.18g/mol 2.48g in

5M NaOH

40g/mol 1M=40g/l 5M = 200g/l 1kg/5L

In the fume hood（在通风橱中操作）

- Put benchcoat paper with absorbant side up

- 将桌布（吸水的一面向上）滤纸铺在实验台上

- Seeds (well dried) in 50ml falcon (10.000 seeds = 250mg)

- 0.5g种子（干燥）放入100ml锥形瓶中（20.000 seeds = 0.5g）

- (ON in water, gently moving. Imbibition of water, so that they will take up the

EMS in water)

- 加入40ml 100mM phosphate buffer，缓慢振荡，4度培养过夜，使之吸水

- 10-20ml 0.05% triton X, vortex, store 5 min, wash 3x with water

- 弃去phosphate buffer，加入20ml 0.05% triton X，振荡5 min，用水洗3次

- 30µl EMS in 10ml KHPO4 buffer, wrap the cap with parafilm and vortex carefully

(EMS settles to the bottom)

- 加入20ml KHPO4 缓冲液 + 60µl EMS，保鲜膜包裹锥形瓶，小心振荡（EMS 沉入瓶

底）

- Rinse tip in 5M NaOH and discard it in the solution

- 用5M NaOH清洗枪头，并把枪头弃入溶液中

- Transfer to the tube containing seeds using a pipette, gently pipette the seeds up

an down,

- 用巴斯德移液器上下缓慢地吹吸锥形瓶，使之混匀

- wrap in parafilm, gently shake for 7.5 hours

- 用保鲜膜包住锥形瓶，室温缓慢振荡7.5小时（磁力搅拌器）

- Rinse pasteur pipette with NaOH and discard it in the solution

- 用NaOH清洗巴斯德移液器，并弃入溶液中

- Lets seeds precipitate, discard EMS in NaOH

- 使种子沉淀，吸取EMS弃入NaOH中

- Take another pipette and wash 2x 15min with 15ml 100mM sodium thiosulphate

(Na

2

S

2

O

3

)

- 取新的移液器吸取30ml 100mM Na

2

S

2

O

3

洗两次（每次15min）

- 4x wash with 20-30ml water, mix, precipitate, discard in NaOH solution (2x)

- 用60ml水洗8（20）次，混匀，静置沉淀，弃入NaOH 溶液中（最后一次清洗时，使

种子浸泡水中1小时，为了让EMS从种子中充分地扩散出来）

- (more) 10ml water, mix 5 min,… (2x)

- 用20ml 水洗两次，混匀5min，重复两次

- Transfer the seeds to a new falcon and add water, gently shake ON (make sure

they are covered with water so they don’t dry out) (4 degrees)

- 转移种子至一新的falcon 中，加入适量水，缓慢摇动（保证种子完全浸泡在水中，不会

变干）（4度）

- (dry seeds on whatman to preserve them)

- 把种子放在沃特曼滤纸上，干燥保存（不需要此步操作）

- After EMS mutagenesis, seeds should be planted immediately in soil or on plates

(orange soil +eventuell ¼ witte korrels)

- EMS 诱变后，种子应立即播在土壤或琼脂糖平板上（园艺土壤或者）

- Discard gloves in 5M NaOH, use pasteur pipette to push them into solution

- 将手套弃入5M NaOH，用巴斯德移液器推进溶液中

- Put on new gloves and clean material with NaOH, then rinse with water

(running water for 30min)

- 戴上新的手套，用NaOH清洗试验台等接触过的仪器设备，然后用流水清洗30min

- Glassware and disposable tips ON in NaOH, put in 2 plastic bags, seal them and

discard with the chemical waste

- 玻璃器皿和一次性墙头放入NaOH溶液中，放在两层塑料袋中，密封，和化学废弃物一

起丢掉

- Everything that may have touched EMS must be soaked in the 5M NaOH for 2

days

- 任何可能接触到EMS的物品必须在5M NaOH浸泡两天

- Let op bij afgieten, EMS is volatile!

- 排水时注意：EMS是挥发物质！！

- Water avond ervoor; water in tray

- 提前一天使土壤吸好水

- Zaaien; oplossen in 0,1% agarose

- 播种：溶解于0.1% 琼脂糖中

- 2ml/ pot (50 zaden/ 2ml)

- 2ml 琼脂糖/盒（50种子/2ml）

- Also sow non-mutagenized seeds/non-treated seeds（同时播种未突变的种子）

- 200 potten（200盒）

o 9 potten/ tray（9盒/托盘）

o 6 trays/ schap（6托盘/架）

o 4 schappen（4货架）

- Na zaaien besproeien en afdekken met plastic（播种和盖上保鲜膜）

200x 50 =10.000 seeds

9 x 6 x 4 = 216 potten