General Protocols Dr,Xue’s lab

Mini petunia DNA Extraction

  1. One of petunia leaf was taken and put in 1.5ml of EP tube, put the tube in liquid nitrogen, ground the leaf with liquid nitrogen and then add 500 (700)ul of CTAB buffer and mix well;
  2. Incubate the tube at 65 of water bath for 30 min;
  3. Add same volume of chloroform:isoamyl alcohol(24:1) and mix gently;
  4. 5 min later, centrifuge the mixture at 12,000 rpm for 5 min;
  5. Transfer the supernatant to a new tube , and add equal volume of isopropanol, mix gently;
  6. 5 min later, the genome DNA can be seen, centrifuge the tube at 10,000 rpm for 2 min;
  7. Carefully discard the supernatant, wash the pellet with 70% ethanol, then air dry;
  8. Dissolve the pellet with 30-50 ul of TE.

CTAB Buffer preparation : (50ml)

CTAB 1.0 g

5M NaCl 14 ml

2-Mercaptoethanol 100 ul

0.5 M EDTA 2 ml (PH 8.0)

1M Tris 5 ml (PH 8.0)

SDW 29 ml

MAX Petunia DNA Extraction

  1. 0.5-1.0 g of petunia leaves were collected and ground in liquid nitrogen;
  2. Add 3ml/g of CTAB buffer and mix well;
  3. Incubate the mixture in 65 of water bath for 30 min;
  4. Add same volume of chloroform:isoamyl alcohol(24:1) and mix well;
  5. Centrifuge the mixture at 4000 rpm for 3 min;
  6. The supernatant was transferred to a new tube and same volume of isopropanol was added and mix well;
  7. Centrifuge the genomic DNA at 10,000 rpm for 10 min, discard the supernatant and wash the pellet with 70% ethanol, then air dry
  8. Resuspended the pellet in TE and store at –20.

Procedure for Tabacco(mini)

  1. Leaves were collected from plants at the six-to-seven leaf stage and grounded in liquid nitrogen;
  2. Add 500 ul of extraction buffer and were put at 65 water bath after mix for 2 min;
  3. Add same volume of phenol , phenol/chloroform, and chloroform , step by step;
  4. After the last step, transfer the supernatant to a new tube , add same volume of isopropanol and 1/10 volume of NaAc(3M,PH5.8) mix well, then put at –20 for 1 hour;
  5. Centrifuge to collect the pellet and wash the pellet with 70% of ethanol;
  6. Air dry the pellet and dissolve in 50 ul of TE(sdw) contain RNase.

Extraction Buffer

components Final con.

sorbitol 0.14 M

Tris(ph8.0) 0.22 M

EDTA(PH8.0) 0.022 M

NaCl 0.8 M

CTAB 0.8%

SDS 1%

Mini Antirrihinum DNA Extract procedure

  1. Pick up two leaves from the plant and put in 1.5 ml of tube, the tube were put in liquid nitrogen immediately;
  2. After the leaves were ground , add 500ul of DNA extraction buffer and mix thoroughly;
  3. Put this tube in water bath at 65 for 2 min(or more), then spin for ,2 min at 12,000 rpm;
  4. Transfer the supernatant to a new tube and mix with same volume of phenol/chloroform(1:1), spin for 5 min at 12,000 rpm;
  5. Transfer the upper part to a new tube, add 1/10 volume of 3M NaAc(PH 5.8) and same volume of isopropanol and mix well, spin at 12,000 for 5 min;
  6. Discard the supernatant, wash the pellet with 70% of Ethanol;
  7. Air dry the pellet and dissolve in 50 ul of sdw or TE(with RNase).

DNA Extraction Buffer

Components Final con.

EDTA(PH 8.0) 50 mM

Tris (PH 8.0) 0.1 M

SDS 1%

NaCl 0.1 M

Yeast Component Cell preparation and Transformation

YPAD Broth (液体培养基)----1L

Peptone 20 g

Yeast extract 10 g

SDW 960 ml

Adjust the PH to 5.8, add 4omg of adenine sulfate, autoclave.

Add 50% of autoclaved glucose 40ml to the final concentration to 2% .

If for plates, add 15-20 g of agar.

注：YEAST 菌种（SP-Q01 S）需储存在-80度，避免反复冻融，以免影响菌的活力。保存在平板上的菌种只能保存一周，若要继续使用，需每周继代一次。

感受态的制备：

1 加1ml的YPAD到1.5ml的管中，接入2-4个酵母单菌落，30度培养（不超过一周），重悬菌液，使其充分混匀；

2 按1：50 的比例接入新鲜的YPAD，30度，225-250rpm摇培18-24小时至OD600达1.2(若超过24小时需重新制备菌液;

3 按1:6的比例加入YPAD(50ml to 300ml), 30c摇培3小时;

4 1000 G 离心5分钟收集细胞,弃上清, 用50ml的去离子水重悬细胞;

5 1000 G 离心5 分钟收集细胞,弃上清, 用1.5ml新鲜的TE-LiAc溶液重悬细胞.

TE-liAc solution(1xTE and 1x LiAc)

1 ml 10 x TE

1 ml 10 x LiAc

8 ml SDW

热击转化

1 制备carrier DNA(salmon spern 20mg/ml),煮20分钟后放入冰上冷却;

2 100ng表达质粒+100ul酵母感受态+100ugcarrier DNA轻轻混匀;

3加入600 ul TE-LiAC-PEG,混匀,30度摇培30分钟(200rpm);

4加入70ul DMSO,轻轻混匀,42度热击20分钟;

5冰上冷却10分钟,3000rpm离心10秒;

6弃上清(尽量除净),加入0.5ml 1xTE,CHX重悬细胞;

7将细胞铺于EMM-thiamine平板上,30度生长4-6天.

TE-LiAc-PEGsolution(1x TE ,1x LiAc, 40%w/vPEG 3350)

1 ml 10x TE

1ml 10x LiAc

8 ml 50%(w/v)PEG 3350

用Trizol提取RNA

1 研磨50-100克材料,用1ml Trizol混匀,室温放置5分钟, 加入200UL氯仿,用力剧烈振荡15秒,室温放置2-3分钟,12000G 4度离心15分钟;

2 将最上层无色溶液(约600UL)移至新管中, 加500UL异丙醇,室温放置10分钟,12000G 4度离心5分钟;

3 去上清,用75%酒精洗沉淀, 空气干燥;

4 用SDW-RNasefree溶解沉淀.

注意: 提取RNA的所有物品均需特殊处理; RNA提取后可放在负80 度保存.

花柱染色观察

1 取花柱于25%的冰醋酸中固定;

2 80度,20分钟;

3Anilineblue染色

4 绿光下观察.

1 Fix styles in FAA for 24hr or more;

2 After rinse in tap water (treat styles in a nearly saturated aqueous solution of NaOH(~8N)for 8-24hr

or cleared by autoclaving in 10%(w/v) aqueous sodium sulphite(is pi,104kpa) for 15min;

3 Transfer the softened styles to a small beaken of tap water for one or more hours to remove most of the sodium hydroxide(sodium sulphite);

4 Stain the styles in a 0.1% solution of aniline blue dye in 0.1N K3PO4 for 1-24 hr(adequate staining can also be done in a small amount of the dye on a glass slide);

5 For observations the styles are mounted in a few drops of the staining medium on clean glass slides and are covered with cover slips;

6 Observe the styles by directing illuminating the slide with UV of ~356 nm in a darkroom.

FAA :( 10ml ) Formalin 1ml + 80%Alchol 8 ml + Acetic acid 1 ml

Aniline Blue: 0.1% aniline blue dissolved in 0.1N K3PO4

7g(3.5g)/L tribasic potassium phosphate

1g/L aniline blue

dissolve in a small quantity of distilled water and mix the solutions and make up to 1 liter.

10% sodium sulfite: 10g of sodium sulfite in 100ml of water

8N NaOH: 32g of NaOH in 100ml of water

花粉萌发

将花粉收集后置于花粉萌发液中,温度为26度,湿度100%,选取不同时间点镜下观察.(一般至少在40分钟以上).

花粉萌发液的配置(有两种):

  1. 硝酸钾-0.01% 2 CaCl2 – 1mM (0.011g/100ml)

硼酸 – 0.01% H3BO4- 1mM (0.0062g/100ml)

硫酸镁-0.02% MgSO4-1mM (0.0246g/100ml)

硝酸钙-0.03% KNO3 – 1mM (0.0101g/100ml)

蔗糖—15% sucrose- 0.25M

PH to 5.6