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Hi-Tail PCR to identify the flanking DNA sequence of T-DNA insertion

Author: Yujiao Peng, updated date: , view: 82, Q&A: 0

Procedure

Hi-Tail PCR 克隆T-DNA 侧翼序列

 

1.    PCR 所用引物(此引物根据pTFCM载体设计)

引物    编号

序列

LB1       PB23

LB2       PB24

LB3       PB25

RB1a      PB26

RB2a      PB27

RB3a      PB28

LAD1     PB18

LAD2     PB19

LAD3     PB20

LAD4     PB21

AC1      PB22

TTTCTCCATA   ATAATgTgTg AgTAgTTCCC AgAT

ACgATggACT CCAgTCCggC CgggTTTCgC TCATgTgTTg AgCATATAAg

CgTTAATTCA gTACATTAAA AACgTCCgCA AT

ggCAATAAAg TTTCTTAAgA TTgAATCCTg T

ACgATggACT CCAgTCCggC CTgTTgCCgg TCTTgCgATg ATTATCA

gTAATgCATg ACgTTATTTA TgAgATgggT T

ACg ATg gAC TCC AgA gCg gCC gC(g/C/A) N(g/C/A)N NNg gAA

ACg ATg gAC TCC AgA gCg gCC gC(g/C/T) N(g/C/T)N NNg gTT

ACg ATg gAC TCC AgA gCg gCC gC(g/C/A) (g/C/A)N(g/C/A) NNN CCA A

ACg ATg gAC TCC AgA gCg gCC gC(g/C/T) (g/A/T)N(g/C/T) NNN Cgg T

ACg ATg gAC TCC AgA g

 

 

 2.各引物的位置

 

 

 

 

 

 

3.PCR扩增(共扩三次)

 

用CTAB法提取转化子的DNA为第一次扩增的模板(将浓度稀释至20-30ng/ul),之后的的第二次和第三次扩增均以上一次对应编号反应体系(即2-1的模板是1-1的PCR产物)的PCR产物经稀释后为模板。

 

第一次扩增(共四个反应):

  第一个反应体系(1-1):      第二个反应体系(1-2):         第三个反应体系(1-3):        第四个反应体系(1-4):

  10×PCR buffer   2ul        10×PCR buffer 2ul        10×PCR buffer  2ul          10×PCR buffer   2ul

2.5mMdNTP    1.6ul         2.5mMdNTP     1.6ul         2.5mMdNTP   1.6ul            2.5mMdNTP  1.6ul

 5uMPB18         4ul           5uMPB19     4ul           5uMPB18      4ul             5uMPB19      4ul

5uMPB20         4ul           5uMPB21     4ul           5uMPB20      4ul             5uMPB21      4ul

 5uMPB26       1.2ul         5uMPB26     1.2ul           5uMPB23     1.2ul            5uMPB23    1.2ul

  Extaq       0.1ul          Extaq      0.1ul           Extaq      0.1ul             Extaq       0.1ul

(20-30ng/ul)DNA     1ul      (20-30ng/ul)DNA  1ul     (20-30ng/ul)DNA    1ul        (20-30ng/ul)DNA    1ul

H2O          6.1ul          H2O        6.1ul         H2O          6.1ul              H2O        6.1ul

20ul                      20ul                        20ul                         20u

 

 

第一次扩增的程序:

step

Temperature(℃)

Time(min:s)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

93

95

94

60

72

Go to step3

94

25

Ramping to 72

72

94

58

72

Go to step11

72

End

2:00

1:00

0:30

1:00

3:00

10times

0:30

2:00

0.5℃/s

3:00

0:20

1:00

3:00

25 times

5:00

 

  

 

 

 

 

 

 

 

第二次扩增(共四个反应):

 

将第一次的PCR产物稀释40倍为模板

 

第一个反应体系(2-1):      第二个反应体系(2-2):         第三个反应体系(2-3):        第四个反应体系(2-4):

10×PCR buffer 2.5ul      10×PCR buffer 2.5ul        10×PCR buffer 2.5ul          10×PCR buffer 2.5ul

2.5mMdNTP    1.6ul        2.5mMdNTP    1.6ul          2.5mMdNTP     1.6ul           2.5mMdNTP     1.6ul

5uMPB22       1.2ul           5uMPB22  1.2ul           5uMPB22     1.2ul             5uMPB22     1.2ul

5uMPB27       1.2ul           5uMPB27   1.2ul           5uMPB24     1.2ul            5uMPB24      1.2ul

Extaq       0.12ul            Extaq   0.12ul           Extaq      0.12ul            Extaq       0.12ul

DNA          1ul               DNA    1ul                DNA       1ul             DNA          1ul

H2O      17.4 ul              H2O    17.4ul         H2O         17.4ul              H2O       17.4ul

25ul                      25ul                        25ul                          25ul

 

 

 

第二次扩增的程序:

step

Temperature(℃)

Time(min:s)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

94

65

72

Go to step1

94

68

72

94

68

72

94

50

72

Go to step5

72                                                   

End

0:20

1:00

3:00

1time

0:20

1:00

3:00

0:20

1:00

3:00

0:20

1:00

3:00

13times

5:00

 

 

­­­­­­­­­­­­­­­­

 

  

 

 

 

 

 

第三次扩增(共四个反应):

 

将第二次的PCR产物稀释10倍为模板

 

第一个反应体系(3-1):      第二个反应体系(3-2):         第三个反应体系(3-3):        第四个反应体系(3-4):

  10×PCR buffer 2.5ul      10×PCR buffer 2.5ul        10×PCR buffer 2.5ul          10×PCR buffer 2.5ul

2.5mMdNTP        1.6ul        2.5mMdNTP     1.6ul       2.5mMdNTP      1.6ul         2.5mMdNTP       1.6ul

 5uMPB22       1.2ul            5uMPB22  1.2ul           5uMPB22    1.2ul             5uMPB22     1.2ul

5uMPB28       1.2ul           5uMPB28   1.2ul           5uMPB25    1.2ul            5uMPB25      1.2ul

 Extaq       0.1ul          Extaq      0.1ul           Extaq      0.1ul            Extaq        0.1ul

DNA        1ul              DNA        1ul             DNA       1ul               DNA          1ul

H2O        17.4 ul          H2O        17.4ul         H2O        17.4ul              H2O       17.4ul

25ul                      25ul                        25ul                          25ul

 

 

 

第三次扩增的程序:

step

Temperature(℃)

Time(min:s)

1

2

3

4

5

6

7

8

9

10

11

12

94

68

72

94

68

72

94

50

72

Go to step1

72                                                   

End

0:20

1:00

3:00

0:20

1:00

3:00

0:20

1:00

3:00

6-7times

5:00

 

 



PtrpC-hyg -TtrpC 片段自pSKHSacI XhoI双酶切后联入同样双酶切的pCAMBIA1301(原始),构建成为pTFCM,转入农杆菌EHA105 (Str/Rif) 后用于盾壳霉ZS-1的转化。但后来鉴定好像缺失了一个XbaI 位点。

 

LB1:  5'  AGG GTT CCT ATA GGG TTT CGC TCA TG  3'   63.6

LB2:  5'  CAT GTG TTG AGC ATA TAA GAA ACC CT   3'   58.8

LB3:  5'  GAA TTA ATT CGG CGT TAA TTC AGT    3'   55.1

 

RB1:  5'  GAT TGA ATC CTG TTG CCG GTC TTG  3'     65

RB2:  5'  TGT TGC CGG TCT TGC GAT GAT TAT C  3'   65

RB3:  5'  GCG CAA ACT AGG ATA AAT TAT C  3'        57

 

AD-1:     5’ NTC gA(g/C) T(A/T)T (g/C)g(A/T) gTT 3’
AD-2:     5’ NgT CgA (g/C)(A/T)g ANA (A/T)gA A 3’
AD-3:     5’ (A/T)gT gNA g(A/T)A NCA NAg A 3’
AD-4:     5’ Ag(A/T) gNA g(A/T)A NCA (A/T)Ag g 3’
 
PCR体系:
dNTP 200uM, special primer 0.15uM, AD primer 2uM, Taq 0.8U, glycerol 6%, DMSO 3%.
 
 

PCR扩增验证转化子:扩增的目标片段是质粒上的Gus基因,大小约为1165 bp引物序列如下:

p15'-GACCAAAGCCAGTAAAGTAGAAC-3' 

p25'-CCCACTATCCTTCGCAAGA-3'





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