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农杆菌介导的玉米小斑病菌的遗传转化

Author: 董俊妃, updated date: , view: 158, Q&A: 0

Materials and Reagents

一、试剂的配置:

1.磷酸氢钾缓冲液(K-buffer, PH5.3)(200mL):

45.64g磷酸氢二钾(K2HPO4)加入双蒸水定容至200ml,用H3PO4调PH至5.3,高压蒸汽灭菌。

2.硫酸镁-氯化钠(M-N)(1L):

七水合硫酸镁(MgSO47H2O) 30g,氯化钠(NaCl) 15g加双蒸水定容1L;高压蒸汽灭菌。

3.1%氯化钙(CaCl2) (W/V)(100mL):

1g CaCl2.H2O加入双蒸水定容100ml;高压蒸汽灭菌。

4.20%葡萄糖(Glucose) (W/V)(100mL):

20g Glucose加入无菌水定容100ml,细菌过滤器过滤灭菌。

5.50%甘油(Glycerol)(100mL):

50ml 100%的Glycerol加入双蒸水定容100ml;高压蒸汽灭菌。

6.1%FeSO4(100mL):

0.01g的FeSO4加入无菌水定容100ml,细菌过滤器过滤灭菌,现用现配。

7.0.1M的乙酰丁香酮(AS):

取196.2mg AS,用二甲亚砜(DMSO)溶解,定容至10ml,即为0.1M AS,将其分装于1.5ml离心管中,-20℃贮存备用,使用前无须灭菌。

8.1M的2-(N-吗啉)乙磺酸钠(MES):

称取7.812gMES,用40mL无菌水完全溶解,调pH为5.3,细菌过滤器过滤灭菌,配成-20℃贮存备用。

9.100mg/ml头孢霉素:

称取100mg头孢霉素(公司:Biosharp)溶解1ml的灭菌双蒸水中,用细菌过滤器过滤除菌,配成100mg/ml溶液装于1.5ml无菌离心管中,将其置于-20℃贮存备用。

10.Spore element(1L)

ZnSO4·7H2O 100mgCuSO4·5H2O 100mgMnSO4·5H2O 100mgNa2MnO4·5H2O 100mgH3BO3 100mg,双蒸水定容到1L,高压蒸汽灭菌。

11. 20%(NH4)2NO3 (w/v)

20g (NH4)2NO3加入双蒸水定容100ml。

12.诱导培养基(IM)(1L):

K-buffer (PH5.3)

10ml

M-N

20ml

1%CaCl2·2H2O(W/V)

1ml

20%(NH4)2NO3(w/v)

2.5ml

50%甘油

10ml

Spore element

5ml

分装50 ml /瓶,高压蒸汽灭菌

用之前加500uL20%葡萄糖,100uL0.1M AS2.5ml1M MES,5uLFeSO4

13.共培养培养基(CO-IM)(1L):

K-buffer (PH5.3)

10ml

M-N

20ml

1%CaCl2·2H2O(W/V)

1ml

20%(NH4)2NO3(w/v)

2.5ml

50%甘油

10ml

Spore element

5ml

Agar

15g

分装200 ml /瓶,高压蒸汽灭菌

用之前加1000uL20%葡萄糖,800uL0.1M AS8ml1M MES,20uLFeSO4


Procedure

二、实验方法

1)将质粒转入农杆菌EHA105中,挑取单菌落在LB(50ug/ml Rif, 50ug/ml Kan)平板上划线活化;

2) 挑取阳性单菌落与5ml LB(50ug/ml Rif, 50ug/ml Kan)180r/min28℃摇床培养过夜;

3) 农杆菌生长至OD600=1.0,用IM培养基将农杆菌菌液稀释至OD600=0.15180r/min28℃摇床培养5h。离心6000r/min5min,收集农杆菌,并用50ml IM培养基溶解农杆菌;

4) 用灭菌双蒸水冲洗TM17斜面,离心6000r/min5min,收集孢子。用含农杆菌的IM培养基稀释孢子至1×106/ml混匀。在CO-IM平板上铺一层玻璃纸(提前高压蒸汽灭菌),吸取300ul农杆菌与TM17孢子的混合液涂于CO-IM平板上,建议涂4-5CO-IM25℃避光培养3d

5)揭下玻璃纸置于灭过菌的培养皿上,上面倒15mlPDA 30ug/ml潮霉素,500ug/ml头孢霉素),25℃培养2d

6)挑取长出的单菌落至于PDA平板( 30ug/ml潮霉素)上;

7)将在PDA平板( 30ug/ml潮霉素)上正常生长的小斑病菌转接到V8培养基上进行培养。


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