Template:Heidelberg/notebook/rd/week33

week number 33

▼2015-08-10 In vitro Transcription of substrate and labeling RNA

In vitro Transcription:

As DNA templates the PCR (24th July 2015) and the annealed primers (30th July 2015) were used.

All solutions were mixed as indicated in the table:

 

cStock

cFinal

V[µL]

Transcription buffer

10 x

1 x

10

ATP

100 mM

4 mM

4

GTP

100 mM

4 mM

4

CTP

100 mM

4 mM

4

UTP

100 mM

4 mM

4

DTT

1 M

10 mM

1

DMSO

  

5 %

5

DNA Template (PCR 2015-07-24)

 

 

10

T7 RNA Polymerase

2 mg/mL

0,1 mg/mL

5

H2O

  

ad 100 µL

53

Final

    

100

 

  • Reaction was incubated for 3 h at 37 °C
  • After 1.5 h another 2 µL T7 RNA Polymerase were added
  • Addition of 2 µL DNase I and further incubation at 37 °C for 20 min

Substrate RNA purification by precipitation:

  • Sample was mixed with 100 µL of 2 x loading dye and purified over a 10 % PAGE
  • Bands were visualized by UV shadowing and suitable bands were excised
  • RNA was eluted out of the gel using 0.3 M NaAc pH 5.5 in three elution steps
  • Gel parts were filtered of and 2.5 volumes of -20 °C EtOH were added, sample was stored at -20 °C oN to let the RNA precipitate
  • Spin sample at 16,000 g for 30 min, discard the supernatant
  • Washed the pellet twice with 70 % EtOH and dissolved RNA in 20 µL of MQ water

 

▼2015-08-11 DNAzyme Activity Assay with candidates with ATP aptamer

Samples:

  • 10-23 DNAzyme: FS032
  • 7-18 DNAzyme: FS 033
  • 10-23 DNAzyme with ATP aptamer with linker: FS019
  • 7-18 DNAzyme with ATP aptamer with linker: FS027
  • 10-23 DNAzyme with ATP aptamer A: FS017, B: FS018
  • 7-18 DNAzyme with ATP aptamer A: FS025, B: FS026
  • t=0: 7-18 DNAzyme und equal amounts of substrate in water and loading dye

DNAzymes were designed according to Wang, 2002. All were purchased as oligos at Sigma Aldich

Substrate and DNAzyme (FS032 and FS033) was incubated together as follows:

 

cStock

cFinal

V[µL]

Tris HCl ph 7.5

1 M

50 mM

1.25

DNAzyme

10 µM

5 µM

1.25

Substrate

1 µM

200 nM

5

NaCl

1 M

100 mM

2.5

MgCl2

1 M

20 mM

0.5

SDS

20 %

0,01 %

1,25

H2O

 

ad 25 µL

2

Final

 

 

25

 

Substrate and DNAzyme (FS019 and FS027) was incubated together as follows:

 

cStock

cFinal

V[µL]

Tris HCl ph 7.5

1 M

50 mM

1.25

DNAzyme

10 µM

5 µM

1.25

Substrate

1 µM

200 nM

5

NaCl

1 M

100 mM

2.5

MgCl2

1 M

20 mM

0.5

SDS

20 %

0,01 %

1,25

ATP

100 mM

5 mM

1,25

H2O

 

ad 25 µL

12

Final

 

 

25

 

Substrate and DNAzyme (FS032 and FS033) was incubated together as follows:

 

cStock

cFinal

V[µL]

Tris HCl ph 7.5

1 M

50 mM

1.25

DNAzyme A

10 µM

5 µM

1.25

DNAzyme B

10 µM

5 µM

6.25

Substrate

1 µM

200 nM

5

NaCl

1 M

100 mM

2.5

MgCl2

1 M

20 mM

0.5

SDS

20 %

0,01 %

1,25

ATP

100 mM

5 mM

1.25

H2O

 

ad 25 µL

5.75

Final

 

 

25
  • All compounds were mixed except the substrate. The samples were heated up to 95 °C for 5 min and cooled on ice
  • Substrate was added and the reaction was heated up to 95 °C for 1 min and then incubated at 37 °C for 2 h
  • 5 µL DNase I were added and everything was further incubated at 37 °C for ~30 min
  • Samples were mixed with an appropriate volume of 2x loading dye and separated on a 20 % denaturing PAGE, stained with SYBR Gold and imaged on a Typhoon Scanner, GE

Results:

The DNAzymes w/o aptamer cleaved the substrate. The band at 62 bp of the substrate disappears for both DNAzymes and a band at ~40 bp appears. For the DNAzymes with ATP aptamer no clear result could be observed.

Assay will be repeated with and w/o the presence of ATP and further controls.

▼2015-08-13 DNAzyme Activity of DNAzymes with ATP aptamer

 

Samples:

  • 10-23 DNAzyme: FS032
  • 7-18 DNAzyme: FS 033
  • 10-23 DNAzyme with ATP aptamer with linker: FS019
  • 7-18 DNAzyme with ATP aptamer with linker: FS027
  • 10-23 DNAzyme with ATP aptamer A: FS017, B: FS018
  • 7-18 DNAzyme with ATP aptamer A: FS025, B: FS026

DNAzymes were designed according to Wang, 2002. All were purchased as oligos at Sigma Aldich

Stock solutions and conditions:

 

cStock

cFinal

Tris HCl ph 7.5

1 M

50 mM

DNAzyme (A)

10 µM

500 nM

DNAzyme B

10 µM

500 nM

Substrate

1 µM

200 nM

NaCl

1 M

100 mM

MgCl2

1 M

20 mM

SDS

20 %

0,01 %

ATP

100 mM

5 mM

H2O

 

ad 25 µL

 

 

 

Pipetting scheme:

#

 

 

Tris HCl ph 7.5

DNAzyme A

DNAzyme B

Substrate

NaCl

MgCl2

SDS

ATP

H2O

Final

1

FS032

10-23D

2.5

2.5

0

10

5

1

2.5

2.5

24

50

2

FS033

7-18D

2.5

2.5

0

10

5

1

2.5

2.5

24

50

3

FS019

10-23DmLink

2.5

2.5

0

10

5

1

2.5

2.5

24

50

4

FS027

7-18DmLink

2.5

2.5

0

10

5

1

2.5

2.5

24

50

5

FS017+18

10-23D_A+B

2.5

2.5

12.5

10

5

1

2.5

2.5

11.5

50

6

FS025+26

7-18D_A+B

2.5

2.5

12.5

10

5

1

2.5

2.5

11.5

50

7

 

 

2.5

2.5

0

10

5

1

2.5

0

26.5

50

8

 

 

2.5

2.5

0

10

5

1

2.5

0

26.5

50

9

FS019

10-23DmLink

2.5

2.5

0

10

5

1

2.5

0

26.5

50

10

FS027

7-18DmLink

2.5

2.5

0

10

5

1

2.5

0

26.5

50

11

FS017+18

10-23D_A+B

2.5

2.5

12.5

10

5

1

2.5

0

14

50

12

FS025+26

7-18D_A+B

2.5

2.5

12.5

10

5

1

2.5

0

14

50

13

FS032

-10-23D

1.25

1.25

0

0

2.5

0.5

1.25

0

18.25

25

14

FS033

-7-18D

1.25

1.25

0

0

2.5

0.5

1.25

0

18.25

25

15

FS019

-10-23DmLink

1.25

1.25

0

0

2.5

0.5

1.25

0

18.25

25

16

FS027

-7-18DmLink

1.25

1.25

0

0

2.5

0.5

1.25

0

18.25

25

17

FS017+18

-10-23D_A+B

1.25

1.25

6.25

0

2.5

0.5

1.25

0

12

25

18

FS025+26

-7-18D_A+B

1.25

1.25

6.25

0

2.5

0.5

1.25

0

12

25

19

 

Substrate only

1.25

0

0

5

2.5

0.5

1.25

0

14.5

25

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  • All compounds were mixed except the substrate. The samples were heated up to 95 °C for 5 min and cooled on ice
  • Substrate was added and the reaction was heated up to 95 °C for 1 min
  • Afterwards 25 µL of samples 1-12 were stored at -20 °C (t=0)
  • The remaining 25 µL and all other samples were incubated at 37 °C for 1.5 h
  • 12.5 µL of samples 13-19 were taken and stored at -20 °C
  • 5 µL and 2.5 µL DNase I were added to 1-12 and 13-19 and everything was further incubated at 37 °C for ~30 min
  • Samples were stored at -20 °C
  • Samples were mixed with an appropriate volume of 2x loading dye
  • 25µL were loaded and separated on a 20 % denaturing PAGE
  • Gel was stained with SYBR Gold and imaged with a Typhoon Scanner, GE

 

Results:

Non-modified DNAzymes cleaved Substrate, while the candidates that contain an ATP aptamer do not lead to detectable cleavage. 7-18 DNAzyme (non-modified) seems to work better in the absence of ATP.

▼2015-08-15 Aussagekräftiger Titel

In vitro Transcription:

As DNA templates the PCR (24th July 2015) and the annealed primers (30th July 2015) were used.

All solutions were mixed as indicated in the table:

 

cStock

cFinal

V[µL]

Transcription buffer

10 x

1 x

20

ATP

100 mM

4 mM

8

GTP

100 mM

4 mM

8

CTP

100 mM

4 mM

8

UTP

100 mM

4 mM

8

DTT

1 M

10 mM

2

DMSO

  

5 %

10

DNA Template (PCR 2015-07-24)

 

 

20

T7 RNA Polymerase

2 mg/mL

0,1 mg/mL

5

H2O

  

ad 100 µL

106

Final

    

200

 

  • Reaction was incubated for 3 h at 37 °C
  • After 1.5 h another 2 µL T7 RNA Polymerase were added
  • Addition of 5 µL DNase I and further incubation at 37 °C for 20 min

Substrate RNA purification by precipitation:

  • Sample was mixed with 100 µL of 2 x loading dye and purified over a 10 % PAGE
  • Bands were visualized by UV shadowing and suitable bands were excised
  • RNA was eluted out of the gel using 0.3 M NaAc pH 5.5
  • Gel parts were filtered of and 2.5 volumes of -20 °C EtOH were added, sample was stored at -20 °C for 2 h to let the RNA precipitate
  • Spin sample at 16,000 g for 30 min, discard the supernatant
  • Dissolved RNA in ~50 µL of MQ water
  • Concentration was determined with an Nanodrop Spectrometer and the extinction coefficient (calculated with IDT Oligo Analyzer)
    • Substrate RNA: A260: 5.401 à 9.44 µM
    • Labeling RNA: A260: 10.121 à 26,45 µM