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week number 32

▼2015-08-04 DNAzyme Activity Assay with DNase I digest and precipitation

Substrate and DNAzyme was incubated together as follows:

DNAzyme:

  • 10-23 DNAzyme: FS032, purchased at Sigma Aldrich
  • 7-18 DNAzyme: FS 033, purchased at Sigma Aldrich

DNAzymes were designed according to Wang, 2002.

 

 

cStock

cFinal

V[µL]

Tris HCl ph 7.5

1 M

50 mM

1.25

DNAzyme

10 µM

5 µM

12.5

Substrate

10 nM

2 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

 

  • All compounds were mixed except the substrate. The samples were heated up to 90 °C for 5 min and cooled on ice
  • Substrate was added and the reaction was incubated at 37 °C for 1.5 h
  • 2 µL DNase I were added and everything was further incubated at 37 °C for 30 min
  • Afterwards 2.5 µL of 3 M NaAc pH 5.5 were added to a final concentration of 0.3 M and 75 µL ice cold EtOH, sample was stored at -20 °C oN to precipitate the nucleic acid
  • Sample was centrifuged for 30 min at 14,000 g to pellet the precipitate, pellet was washed with 70 % EtOH twice
  • Afterwards it was dried and dissolved in 10 µL MQ H2O and mixed with an appropriate volume of 2x loading dye
  • Samples were separated on a 20 % denaturing PAGE, stained with SYBR Gold and imaged on a Typhoon Scanner, GE

Results:

Amount of DNAzyme was reduced but did not disappear. Amount of RNA was still under the detection limit.

▼2015-08-06 MALDI-TOF-MS of N3 modified HRP

The purified and modified HRP was spotted in a 1:4 ratio with sinapinic acid matrix on an Opti-TOF 384 well MALDI plateand measured on an AB Sciex TOF/TOF 5800 system in linear mode. The mass of the protein could be found but there was no significant shift when comparing modified and unmodified protein.
Outlook:
Samples will be check for modification using an alkyne modified fluorophore and separated on a SDS-PAGE 

▼2015-08-08 N3 HRP click test with FAM-Alkyne

N3 modified HRP was incubated with alkyne FAM:

 

cStock

cFinal

V[µL]

Phosphate buffer pH 7.0

100 mM

50 mM

50

FAM-Alkyne

1 mM

50 µM

5

N3 HRP

1 mM/ 100 µM/ 10 µM/ 1 µM

50 µM/ 5 µM/ 500 nM/ 50nM

5

Cu(II)SO4

20 mM

100 µM

0,5

THPTA

50 mM

500 µM

1

Sodium ascorbate

100 mM

1 mM

1

H2O

  

Ad 100

37,5

Final

  

 

100

Conditions as Winz, 2012.

As negative control N3 HRP w/o alkyne FAM was incubated and also a sample of alkyne FAM w/o N3 HRP was loaded onto the gel.

Results:

N3 modification reaction was probably not that efficient. Only a very faint band could be visualized with fluorescence scan at the correct size. The amount of protein was under the detection limit of silver nitrate staining.

N3 activation has to be repeated.

▼2015-08-09 DNAzyme Activity with inceased amount of substrate

Substrate and DNAzyme was incubated together as follows:

DNAzyme:

  • 10-23 DNAzyme: FS032, purchased at Sigma Aldrich
  • 7-18 DNAzyme: FS 033, purchased at Sigma Aldrich
  • Control: All compounds but substrate, all compound but DNAzyme
  • t=0 the same amount of substrate as in all samples was mixed with water and loading dye

DNAzymes were designed according to Wang, 2002.

 

 

cStock

cFinal

V[µL]

Tris HCl ph 7.5

1 M

50 mM

1.25

DNAzyme

10 µM

5 µM

12.5

Substrate

1 µM/ 100 nM

200 nM/ 20 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

 

  • All compounds were mixed except the substrate. The samples were heated up to 90 °C for 5 min and cooled on ice
  • Substrate was added and the reaction was incubated at 37 °C for 1.5 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 cleave the substrate. The band at 62 bp of the substrate disappears for both DNAzymes and a band at ~40 bp appears a cleavage takes place.