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</blockquote>==藥品配製== 1.胚胎培養液

14 mM NaCl
0.54 mM KCl
0.026 mM Na2HPO4
0.1 mM CaCl2
0.1 mM MgSO4.7H2O

2.色素抑制劑(phenylthiocarbamide)(1-phyeny-z-thiourea, PTU)

stock solution 2mM
↓10x 稀釋
working solution 0.2mM

3. PBS(per liter)

8 mg NaCl
2.88mg Na2HPO4
0.4mg KCl
0.48mg KH2PO4, pH7.4


4. 4% paraformaldehyde/PBS solution(保存期限:1星期)

取paraformaldehyde(存於4℃中),溶於PBS solution中,使其濃度為4%(v/v)。於68℃水溶槽中靜置,待溶解後,保存於-20℃中。

5. proteinase K stock solution

6. yeast tRNA 10mg/ml

7. PBT solution: 0.1% tween20 in PBS

8. HYB-: (pH=5.0)以citric acid (1M or 2M均可)調整pH,設總體積為500ml時:

60% pormamide (300ml)
4x SSC (取100ml 20x SSC)
0.1% Tween (取500μl) Tween怕光,要包錫箔紙。

溶於DEPC-H2O中

9. HYB+: (pH=5.0)以citric acid調pH值。設體積為250ml

60% pormamide (150ml)
4x SSC (取50ml 20x SSC)
0.1% Tween (取250μl) Tween怕光,要包錫箔紙。
50μg/ml Heparin (取12.5mg)
500μg/ml Yeast tRNA (取10mg/ml yeast tRNA 12.5ml)

溶於DEPC-H2O中

預留空間調pH值

10. maleic acid 1M pH7.5

11. citric acid 1M

12. Tris-HCl 1M pH=9.5(以HCl調整)

配100ml:取12.11g tris,加入80ml 3`H2O,以HCl調整pH,把總體積加到100ml

13. MgCl2 1.0M 取20.33g加三次水至100ml

14. NaCl 1.5M 取8.766g加三次水至100ml

15. NaCl 1.0M 取5.844g加三次水至100ml

16. 100mM Maleic acid (3x) 取 300mM maleic acid 100ml

17. 150mM NaCl(10x) 取1.5M NaCl 30ml

18. 19.0.1% Tween 20  取300μl (將17和18混合後,加三次水至總體積為300ml)

20. 4x blocking reagent :取2g blocking reagent 粉末(存於4℃冰箱)加50ml上列混合液。Blocking reagent不易溶解,配置好後經過高壓滅菌,即可溶解完全。

21. 20x SSC (per liter)

175.3mg NaCl
88.2mg NaOAc
adjust to pH=7.0(可省略)
→滅菌

22. NTMT solution

0.1M NaCl
0.1M Tris-HCl(用HCl調pH值)
50mM MgCl2
0.1% Tween 20

探針置備|RIBOPROBE PREP编辑

1. plasmid DNA linearized:

取10μg DNA,以restriction enzyme作用。
經過phenol, chloroform, 酒精沉澱
用DEPC-ddH2O將體積調至10μl

2. 取1μg purified template DNA 以下步驟在冰上進行

Using Roche kit 1 175 025
(1)先加入適當體積的DEPC-ddH2O,使最終體積為20μl
(2)加入template DNA 1μg
(3)加入0.5μl (RNase inhibition)
(4)加入2μl (DIG/flurescin labble mix)
(5)加入2μl (10x buffer)
(6)加入2μl (SP6 orT7)
mix gently, spin down

3. 37℃ 2小時

4. 加入2μl DNase1,37℃ 20分鐘。

5. 加入formamide 80μl。取1μl跑電泳。

6. 保存於-20℃。


實驗步驟编辑

1.收集卵,置於胚胎培養液中,培養於28.5℃備用。

2.第14hr後加色素抑制劑PTU(ex:27ml 3’水加3ml PTU(2mM)最後濃度為0.2mM)

3.將embryo置於tube中(最多30顆)加入4% paraformaldehyde/ PBS solution,置於4℃中12~16小時,或置於室溫2hr

4. 剝除卵膜

5. 脫水 Dehydration

200μl 25% methanol + 75% PBT 5min
200μl 50% methanol + 50% PBT 5min
200μl 75% methanol + 25% PBT 5min
200μl 100% methanol 10min×3

脫水後可保存於-20℃中,約可保存6個月

6. Re-hydration

200μl 75% methanol + 25% PBT 5min
200μl 50% methanol + 50% PBT 5min
200μl 25% methanol + 75% PBT 5min
200μl 1x PBT 5min×4

第4次用以PBT配製的proteinase K處理

7.proteinase K

incubate embryos in 200μl proteinase K at room temperature(RT)
embryo stage Concentration of ProK Time(26℃) min
<24h No need of ProK ---
24h 10ng/mg 12-15
36h 10ng/mg 15-50
48h 25 ng/mg 25
60h 25 ng/mg 30
72h 50 ng/mg 25
84h 50 ng/mg 30
96h 100 ng/mg 25
  • Stop reaction with 200μl PBT
  • re-fix embryos in 200μl 4% paraformaldehyde/PBS for 20min at RT
  • wash embryos with 200μl PBT for 5min 3times.

8.Pre-hybrydization

preheat HYB- to 60℃
incubate embryos in 200μl preheated HYB- at 65℃ for 5min
pre-hybridize embryos in 200μl HYB+ at 65℃for 3hrs

9.Heat RNA probe-HYB+ (each 200μl HYB+ containing 20~100ng RNA probe) at 68℃ for 10min, 快速置於冰上。 Add 500μl RNA probe- into embryos. Incubate embryos at 65℃overnight.

10.Post-hybridization washing

wash embryos with preheated solution

75% HYB-/25% 2x SSCT 65℃ 10min
50% HYB-/25% 2x SSCT 65℃ 10min
25% HYB-/25% 2x SSCT 65℃ 10min
2×SSCT 65℃ 10min
0.2×SSCT 70℃ 45min×2

11.Absorption(anti-dig antibody reaction)

rinse the embryo with 200μl maleic acid buffer(100mM maleic acid, 150mM NaCl, 0.1%Tween 20)then decade.
add 500μl 1xBlocking reagent and stand at RT for 3 hrs.
decade blocking reagent and add 500μl antibody (anti-DIG-AP antibody, Roche) incubate at 4℃ overnight
wash embryo with 200μl maleic acid buffer for 30min ×4times.

12. 呈色Coloring

加入新配製的NTMT buffer, 5min, 3times, at RT. (NBT/BCIP在NTMT的pH下最易呈色。故先以NTMT改變embryo的pH值。)
500μl NBT/BCIP containing buffer, 1-2hrs, at RT
stop reaction with quick rinses in PBT for several times. 或10min, 4 times.
wash embryos with methanol for 20min 1~3times at RT (洗去不必要的背景。)
wash embryo with PBS and store in PBS at 4℃.

13.拍照


討論|Discussion编辑


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