Team:Heidelberg/notebook/rtsms

week number 37

▼2015-09-08 31. Analysis of Transcription Efficiency using different Promotors

Aim: Screening different promotors (T7, T7 2.5, T3, E. coli and Sp6) to receive information about transcription efficiency

Procedure:

DNA templates were generated by hybridization of 10 µM of each ssDNA (containing different promotors) at 90 °C
Templates were then cooled down to room temperature

Construct	Description	Forward Primer	Reverse Primer
T7 promotor		MS14	MS15
T7 2.5 promotor		MS23	MS24
T3 promotor		MS27	MS28
E. coli		MS29	MS30
Sp6		MS25	MS26

RNA was renatured in 1x Renaturation Buffer at 90 °C and then cooled down for 5 min

cstock

cfinal

T7

T 7.25

T3

Sp6

E. coli

-

+

-

+

-

+

-

+

-

+

Transcription Buffer with 10 mM Spermidine

10x

1x

6 µL

6µL

6 µL

NTP

100 mM

4 mM

9.6 µL

DTT

1 M

10 mM

0.6 µL

Malachite green

20 mM

100 µM

0.3 µL

DFHBI

20 mM

100 µM

0.3 µL

Glycerol

50 %

0.075 %

3 µL

Malachite Green Aptamer (DNA Template)

10 µM

1 µM

6 µL

Renatured RNA

500 nM

12 µL

Inorganic Pyrophosphatase

0.1 U

0.0017 U

1 µL

RiboLock

40 U

0.46 U

0.7 µL

T7

1 mg/mL

0.05 mg/mL

3 µL

T3

3 µL

Sp6

3 µL

E. coli

3 µL

Millipore water

20.5 µL

- w/o T7 RNA Polymerase

+ with T7 RNA Polymerase

Fluorescence was measured in the Tecan Safire 2 for 12 hours

Conditions: Conditions are listed in assay 25

Results: The data shows that the T7 and T7 2.5 are the fastest RNA polymerase according to their ATP consumption and the increasing signal of Malachite Green Aptamer in the Assay. The T3 RNA Polymerase is slower than both T7 RNA Polymerase, but it is still active after 500 min. The Sp6 has a higher Malachite Green Aptamer fluorescence while having a small ATP consumption in comparison to the T3 RNA polymerase. The E. coli RNA polymerase shows no activity.

▼2015-09-08 32. Titration curve of the T7 and T3 RNA Polymerase

Aim: Developing a model based on the data generated by this assay

Procedure:

RNA and DNA was renatured as described in previous assays

	cstock	cfinal	1		1:2		3		4
Polymerase			T3	T7	T3	T7	T3	T7	T3	T7
Transcription Buffer 1 mM Spermidine	10x	1x	6 µL	6µL	6 µL	6µL	6 µL	6µL	6 µL	6µL
NTP	100 mM	4 mM	9.6 µL	9.6 µL	9.6 µL	9.6 µL	9.6 µL	9.6 µL	9.6 µL	9.6 µL
DTT	1 M	10 mM	0.6 µL	0.6 µL	0.6 µL	0.6 µL	0.6 µL	0.6 µL	0.6 µL	0.6 µL
Malachite green	20 mM	100 µM	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL
DFHBI	20 mM	100 µM	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL
Glycerol	50 %	0.075 %	3 µL	3 µL	1.5 µL	1.5 µL	1 µL	1 µL	2.7 µL	2.7 µL
Malachite Green Aptamer (DNA Template)	10 µM	1 µM	6 µL	6 µL	6 µL	6 µL	6 µL	6 µL	6 µL	6 µL
Renatured RNA (ATP AptamerJAWS1 Spinach2)		500 nM	12 µL	12 µL	12 µL	12 µL	12 µL	12 µL	12 µL	12 µL
Inorganic Pyrophosphatase	0.1 U	0.0017 U	1 µL	1 µL	1 µL	1 µL	1 µL	1 µL	1 µL	1 µL
RiboLock	40 U	0.46 U	0.7 µL	0.7 µL	0.7 µL	0.7 µL	0.7 µL	0.7 µL	0.7 µL	0.7 µL
T7/ T3					3 µL	3 µL	1.5 µL	1.5 µL	0.3 µL	0.3 µL
Millipore water			20.5 µL	20.5 µL	20.5 µL	20.5 µL	20.5 µL	20.5 µL	20.5 µL	20.5 µL

The readout was performed in the Tecan Safire 2

Conditions: Measurement conditions are listed in assay 25

Results: With the decrease of enzyme concentration we can see that the fluorescence of the ATP Aptamer Spinach2 decreases slower than under higher concentrated RNA Polymerase concentration. Hence, the increase of the Malachite Green Aptamer is slower, too. The 1:10 thinner shows no increase of Malachite Green Aptamer Fluorescence and slight decrease of the ATP Aptamer Spinach2 fluorescence.

▼2015-09-08 33. Comparison of the Spectra of all ATP Aptamer Spinach2 Variations

Aim: Demonstration of the enhancement of the ATP Aptamer Spinach2 made by the software JAWS by exchanging the stem

Procedure:

RNA was renatured in 1x Renaturing buffer
The setup is listed in assay protocol 13

Conditions: The Spectra were measured in the JASCO. Conditions are shown in experiment 14

Results: The results show that the computer enhanced ATP Aptamer Spinach2 has a higher fluorescence than the Szostak ATP Aptamer that we added to the Spinach2. By comparing the ATP AptamerJAWS1 Spinach2 to the ATP AptamerJAWS2 Spinach2 we can see that the ATP AptamerJAWS2 Spinach2 has a higher peak than the ATP AptamerJAWS1 Spinach2.

▼2015-09-09 34. Miniprep of the RFC Construct containing Spinach2

Aim: Purification of the Plasmid pSB1C3 containing the Spinach2

Procedure:

Overnight culture of the E. coli containing the Spinach2 RFC in a LB Medium with Chloramphenicol
Miniprep was performed using the QIAprep Spin Miniprep Kit

Results: The plasmid pSB1C3 was successfully purified and can be used for further applications.

▼2015-09-10 35. EcoRV Digest of the BioBrick pSB1C3

Aim: Extraction of the BioBrick using the EcoRV digest of the pSB1C3

Procedure:

The purified pSB1C3 was digested with 1x EcoRV Buffer (NEB) and 5 µL Eco RV HF (NEB ) for 1 hour at 37 °C
The digest was analyzed on a 2 % agarose gel

Results: On the gel we can observe two bands on the. The construct is just visible as a streak at 200 bp.

▼2015-09-11 36. Amplifying the FS Substrate 72, the RFC Construct containing Spinach2 and BioBricks Hammerhead Ribozyme with Malachite Green, ATP AptamerJAWS1 Spinach2 and ATP AptamerJAWS2 Spinach2 using the Polymerase Chain Reaction

Aim: Amplification of the fragments for different applications

Procedure:

Assay has been performed as described in experiment 9
Primers that were used:

	Forward Primer	Reverse Primer
FS Substrate 72	FS Substrate 72 PCR Product from Frieda)	MS 42 T7 Promotor MS 44 Rev Substrate HHR MGA (A) MS 45 Rev Substrate HHR MGA (B) MS 46 Rev Substrate HHR MGA (C)
Hammerhead Ribozyme	MS 31 Malachitgreen fwd Primer	MS 32 Malachite green rev Primer
ATP Aptamer Spinach 2/12	MS 37 fwd 12 Spinach ATP	MS 39 rev 12 Spinach ATP
ATP Aptamer Spinach 2/13	MS 38 fwd 13 Spinach ATP	MS 40 13 rev Spinach ATP
pSB1C3 containing

Conditions were used as listed in experiment 9

Analytical Agarose Gel:

2 % Agarose
70 mL TBE
2 µL Ethidium Bromide

Results: DNA was successfully generated. For the FS Substrate, we had to do a gel extraction using the QIAquick Gel Extraction Kit. Hence, the PCR of the FS Substrate was repeated only using the MS 42 T7 Promotor, the MS 46 rev Substrate and the extracted DNA fragment.

▼2015-09-11 37. Purification of Fragments Amplified by PCR using a Purification Kit

Aim: Preparation of the DNA template for in vitro transcription and the cloning of BioBricks

Procedure:

PCR was purified by using the Quiagen PCR-Purification Kit
DNA was eluted form the column with 50 µL Millipore water

Result: DNA was purified and is ready for the in vitro transcription and for the cloning into our RFC standard.

▼2015-09-11 38. Monitoring of the Increase of Fluorescence of the RFC containing Spinach2 and the purified PCR Product in a 384 Well Micro Plate Format during in vitro Transcription

Aim: Proof of concept that the RFC is containing a functional RNA that is purified by T7 RNA Polymerase

Procedure:

	cStock	cfinal	Blank	Functional Setup with Heparin (1)	Functional Setup with Heparin (2)
Transcription Buffer with 10 mM Spermidine	10x	1x	6 µL	6 µL	6 µL
NTP	100 mM	4 mM	9.6 µL	9.6 µL	9.6 µL
DTT	1 M	10 mM	0.6 µL	0.6 µL	0.6 µL
DFHBI	20 mM	100 µM	0.3 µL	0.3 µL	0.3 µL
Glycerol	50 %	0.075 %	3 µL
PRC Product of the RFC			6 µL	6 µL
pSB1C3					6 µL
Inorganic Pyrophosphatase	0.1 U	0.0017 U	1 µL	1 µL	1 µL
RiboLock	40 U	0.46 U	0.7 µL	0.7 µL	0.7 µL
T7	1 mg/mL	0.05 mg/mL		3 µL	3 µL
Millipore Water			32.8 µL	26.8 µL	26.8 µL

Assay was measured in the Tecan Safire 2

Conditions: Conditions are listed in assay 25

Results: We can observe the increase of fluorescence over time which is caused by the increase of the Spinach2 RNA concentration that binds to the DFHBI.

▼2015-09-12 39. Testing the FS Substrate 72 on different Buffer Conditions during in vitro Transcription

Aim: Showing the optimization of the in vitro Transcription for the FS Substrate 72 by testing different buffers. Furthermore we will employ a hammerhead ribozyme for the cleavage of substrate from the Malachite Green Aptamer

Procedure:

DNA template was amplified in protocol 36
RNA was renatured in 1x Renaturing buffer
DNA and RNA was used for the following setup

cStock

cfinal

Blank 1

DNA template with 10 mM Spermidine

Blank 2

DNA template with 1 mM Spermidine

Transcription Buffer with 10 mM Spermidine

10x

1x

6 µL

Transcription Buffer with 1 mM Spermidine

10x

1x

6 µL

NTP

100 mM

4 mM

9.6 µL

DTT

1 M

10 mM

0.6 µL

Malachite green

20 mM

100 µM

0.3 µL

DFHBI

20 mM

100 µM

0.3 µL

Glycerol

50 %

0.075 %

3 µL

FS Substrate 72 with Malachite Green Aptamer

6 µL

Renatured RNA		500 nM	12 µL	12 µL	12 µL	12 µL
Inorganic Pyrophosphatase	0.1 U	0.0017 U	1 µL	1 µL	1 µL	1 µL
RiboLock	40 U	0.46 U	0.7 µL	0.7 µL	0.7 µL	0.7 µL
T7 RNA Polymerase	1 mg/ mL	0.05 mg/ mL		3 µL		3 µL
Millipore Water			20.5 µL	20.5 µL	20.5 µL	20.5 µL

The readout was performed in the Tecan Safire 2
Samples were analyzed on an analytical denaturing polyacrylamide gel (10 % polyacrylamide) stained with Sybr Gold (Invitrogen)

Conditions: All parameters are listed in experiment 25

Results: The experiment shows that we just get slow increase of the fluorescence of the Malachite Green Aptamer; whereas the concentration of the ATP decreases rapidly. On the gel we were able to observe the cleaved Malachite Green Aptamer from the RNA of Interest.

week number 36

▼2015-09-03 27. Real time Monitoring allows the Adjustment of Buffer Conditions for in vitro transcription

Aim: Identification of the best buffer conditions for in vitro transcription by using this big screening method. Buffers differ in pH, Buffer system (Tris-HCl or HEPES) or in the spermidine concentration.

Procedure:

cstock

cfinal

1

2

3

4

5

6

-

+

-

+

-

+

-

+

-

+

-

+

Transcription Buffer w/o Spermidine

10x

1x

6 µL

6µL

Transcription Buffer with 10 mM Spermidine

10x

1x

6 µL

6µL

Transcription Buffer with 100 mM Spermidine

10x

1x

6 µL

6µL

HEPES Transcription Buffer

5x

1x

6 µL

6µL

Tris HCl Transcription Buffer

10x

1x

6 µL

6µL

BSA Transcription Buffer

10x

1x

6 µL

6µL

NTP

100 mM

4 mM

9.6 µL

DTT

1 M

10 mM

0.6 µL

Malachite green

20 mM

100 µM

0.3 µL

DFHBI

20 mM

100 µM

0.3 µL

Glycerol

50 %

0.075 %

3 µL

Malachite Green Aptamer (DNA Template)

10 µM

1 µM

6 µL

Renatured RNA

(ATP Aptamer Spinach 2/12)

500 nM

12 µL

Inorganic Pyrophosphatase

0.1 U

0.0017 U

1 µL

RiboLock

40 U

0.46 U

0.7 µL

T7

1 mg/mL

0.05 mg/mL

3 µL

Millipore water

20.5 µL

- w/o T7 RNA Polymerase

+ with T7 RNA Polymerase

Conditions: The assay was performed in the Tecan Safire 2 under the conditions describes in protocol 25.

Results: The data shows that the transcription buffers generate the biggest yield and the highest ATP consumption. The Tris HCl buffer and the BSA buffer also show an increase of Malachite Green Aptamer and a decrease of the fluorescence of the ATP Spinach2. But both signals are less steep than the changes of signal in the transcription buffers. The HEPES buffer shows no signal. Hence, we expect no transcribed RNA.

▼2015-09-04 28. Titration Curve of the Malachite Green Aptamer

Aim: Determination of the connection between Malachite Green Aptamer concentration and RFU

Procedure:

Malachite Green Aptamer with T7 promotor (generated from MS14 and MS15) were transcribed in vitro according to assay 22
The transcribed RNA was purified by a denaturing polyacrylamide gel electrophoresis
The in 0.3 M sodium acetate eluted RNA was precipitated using isopropanol and centrifuged at 14. 000 g for 1 h 30 min
The RNA was purified in a Amicon Ultra 0.5 mL with a 3 kDa colum which was centrifuged at 10.000 g for 30 min
After each centrifugation, the RNA was washed with 200 µL millipore water
Repeat the step 5 times
Resuspend the RNA in 500 µL millipore water
Measure the concentration of the RNA using a nanodrop:

	Malachite Green Aptamer RNA
Concentration	53.97 µM

Measurement of Malachite Green Aptamer Fluorescence:

Assay was performed with following concentration:

	cStock	cfinal	Blank	1	2	3	4	5	6
Transcription Buffer with 10 mM Spermidine	10x	1x	6 µL	6 µL	6 µL	6 µL	6 µL	6 µL	6 µL
NTP	100 mM	4 mM	9.6 µL	9.6 µL	9.6 µL	9.6 µL	9.6 µL	9.6 µL	9.6 µL
DTT	1 M	10 mM	0.6 µL	0.6 µL	0.6 µL	0.6 µL	0.6 µL	0.6 µL	0.6 µL
Malachite green	20 mM	100 µM	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL
DFHBI	20 mM	100 µM	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL
Glycerol	50 %	0.075 %	3 µL	3 µL	3 µL	3 µL	3 µL	3 µL	3 µL
Renatured RNA of the Malachite Green Aptamer	53.97 µM	10 µM		3.7 µL
		7.5 µM			2.8 µL
		5 µM				1.8 µL
		2.5 µM					0.9 µL
	5.397 µM	1 µM						1.8 µL
	5.397 µM	0.5 µM							0.9 µL
Inorganic Pyrophosphatase	0.1 U	0.0017 U	1 µL	1 µL	1 µL	1 µL	1 µL	1 µL	1 µL
RiboLock	40 U	0.46 U	0.7 µL	0.7 µL	0.7 µL	0.7 µL	0.7 µL	0.7 µL	0.7 µL
Millipore Water			38.5 µL	34.8 µL	35.7 µL	36.7 µL	37.6 µL	36.7 µL	37.6 µL

The fluorescence was measured in a TECAN SAFIRE 2

Conditions: Parameters are shown in assay 25

Results: With the data (n=9) we were able to design a titration curve with an R2=0.997. Using this titration curve, we are able to predict the concentration of the transcribed RNA within our assay. This can be used to model the reaction.

▼2015-09-04 29. Inhibition of in vitro Transcription using Heparin as an Inhibitor

Aim: Showing the reduction of transcribed RNA in presence of an T7 RNA Polymerase inhibitor

Procedure:

RNA and DNA were renatured as described in assay 25

	cStock	cfinal	Blank	Functional Setup with Heparin (1)	Functional Setup with Heparin (2)
Transcription Buffer with 10 mM Spermidine	10x	1x	6 µL	6 µL	6 µL
NTP	100 mM	4 mM	9.6 µL	9.6 µL	9.6 µL
DTT	1 M	10 mM	0.6 µL	0.6 µL	0.6 µL
Malachite green	20 mM	100 µM	0.3 µL	0.3 µL	0.3 µL
DFHBI	20 mM	100 µM	0.3 µL	0.3 µL	0.3 µL
Glycerol	50 %	0.075 %	3 µL		3 µL
Malachite Green Aptamer (DNA Template)	10 µM	1 µM	6 µL	6 µL	6 µL
Renatured RNA		500 nM	12 µL	12 µL	12 µL
Heparin	40 mg/mL	0.7 mg/mL		1 µL
Heparin	40 mg/mL	1.3 mg/mL			2 µL
Inorganic Pyrophosphatase	0.1 U	0.0017 U	1 µL	1 µL	1 µL
RiboLock	40 U	0.46 U	0.7 µL	0.7 µL	0.7 µL
T7	1 mg/mL	0.05 mg/mL		3 µL	3 µL
Millipore Water			20.5 µL	19.5 µL	18.5 µL

Conditions: The samples were measured in the Tecan Safire. Parameters were used as listed in assay 25

Results: The data shows that there is no in vitro transcription in presents of both concentrations of heparin. Therefore the ATP Aptamer Spinach2 and the Malachite Green Aptamer signal are constant. No changes have been observed over time.

▼2015-09-05 30. Evaluating the impact of different DNA template concentrations

Aim: Using the titration curve that was established in the previous assay to say how much DNA of different concentrations is transcribed in the experiment. This assay should help to understand if there is a limit of DNA concentration which can be transcribed or not.

Procedure:

DNA template and ATP AptamerJAWS1 were renatured as described as in

	cStock	cfinal	Blank	1	2	3
Transcription Buffer with 10 mM Spermidine	10x	1x	6 µL	6 µL	6 µL	6 µL
NTP	100 mM	4 mM	9.6 µL	9.6 µL	9.6 µL	9.6 µL
DTT	1 M	10 mM	0.6 µL	0.6 µL	0.6 µL	0.6 µL
Malachite green	20 mM	100 µM	0.3 µL	0.3 µL	0.3 µL	0.3 µL
DFHBI	20 mM	100 µM	0.3 µL	0.3 µL	0.3 µL	0.3 µL
Glycerol	50 %	0.075 %	3 µL
Malachite Green Aptamer	50 nM	0.83 nM		1 µL
		1.67 nM			2 µL
		2.5 nM				3 µL
Renatured RNA		500 nM	12 µL	12 µL	12 µL	12 µL
Inorganic Pyrophosphatase	0.1 U	0.0017 U	1 µL	1 µL	1 µL	1 µL
RiboLock	40 U	0.46 U	0.7 µL	0.7 µL	0.7 µL	0.7 µL
T7 RNA Polymerase	1 mg/ mL	0.05 mg/ mL		3 µL	3 µL	3 µL
Millipore Water			26.5 µL	25.5 µL	24.5 µL	23.5 µL

Fluorescence was measured in the Tecan Safire 2

Conditions: Parameters are listed in assay 25

Results: This assay was used to quantify the Malachite Green Aptamer RNA in comparison to the input DNA. The results show the more DNA the more RNA you get.

week number 35

▼2015-08-25 20. Amplification of ATP AptamerJAWS1 Spinach2 and ATP AptamerJAWS2 Spinach2 via Polymerase Chain Reaction

Aim: Generating a DNA template that contains optimized ATP Aptamer Spinach2, developed by the software JAWS. This template should be used for further fluorescence measurements.

Procedure:

Assay has been performed in a set up described in experiment 14
Primers that were used: Forward Primer - the universal Spinach2 primer (MS01) and the additional reverse primers (MS12 for ATP AptamerJAWS1 Spinach2 and MS13 ATP AptamerJAWS2 Spinach2)
Conditions were used as listed in experiment 14

Analytical Agarose Gel:

2 % Agarose
70 mL TBE
2 µL Ethidium Bromide

Results: DNA was successfully generated and has an expected length of 168 bp.

▼2015-08-25 21. Purification of both ATP Aptamer Spinach2 Constructs via Purification Kits

Aim: Preparation of the DNA template for in vitro transcription of ATP Aptamer Spinach2

Procedure:

PCR was purified by using the Quiagen PCR-Purification Kit
DNA was eluted form the column with 100 µL Millipore water

Results: DNA was purified and is ready for the in vitro transcription.

▼2015-08-25 22. In vitro Transcription and Purification of the ATP Aptamer Spinach2 variations

Aim: Transcription of DNA into RNA that will be used in further assays

Procedure:

	cStock	Cfinal	Volume [µL]
Transcription Buffer	10x	1x	100
DTT	1 M	10 mM	10
ATP	100 mM	4 mM	40
UTP	100 mM	4 mM	40
GTP	100 mM	4 mM	40
CTP	100 mM	4 mM	40
DMSO	100 %	5 %	50
DNA Template (prepared in exp. 21)			100
T7 RNA Polymerase	1 mg/mL	0.05 mg/ mL	50
Millipore Water			530

Conditions:

Incubate for 3 h, 37 °C
Digest the DNA template with 50 U DNase I for 20 min
Purification was achieved by following protocol 7 and 8

Result: In this set up we synthesized and purified the RNA that we will use for the in vitro monitoring assays of ATP.

▼2015-08-26 23. in vitro Monitoring of ATP concentration During Dephosphorylation

Aim: Sensing small molecules in real time by the ATP Aptamer Spinach2 in biochemical reactions. As a biochemical reaction, the reaction of the Apyrase, which converts ATP to AMP and ADP was chosen. A decrease in the fluorescence level of the ATP Aptamer Spinach2 in presence of the Apyrase is expected if the sensor functions well.

Procedure:

	cstock	cfinal	Blank w/o ATP	Blank w/o DFHBI	Functional ATP 1	Functional ATP 2	Functional ATP 3	EDTA Inhibiton of Setup
Renaturing Buffer	2x	1x	20 µL	20 µL	20 µL	20 µL	20 µL	20 µL
ATP AptamerJAWS1 Spinach2		500 nM	8 µL	8 µL	8 µL	8 µL	8 µL	8 µL
CaCl2	100 mM	4 mM	1.6 µL	1.6 µL	1.6 µL	1.6 µL	1.6 µL	1.6 µL
DFHBI	20 mM	100 µM	0.3 µL		0.3 µL	0.3 µL	0.3 µL	0.3 µL
DMSO	100 %	0.75 %		0.3 µL
EDTA	100 mM	6 mM						2.4 µL
ATP	100 mM	1 mM	0.4 µL	0.4 µL	0.4 µL			0.4 µL
	10 mM	200 µM				0.8 µL
	10 mM	100 µM					0.4 µL
Apyrase	50 00 mu/mL	125 mu/mL	1 µL	1 µL	1 µL	1 µL	1 µL	1 µL
Millipore Water			8.7 µL	8.7 µL	8.7 µL	8.3 µL	8.7 µL	6.3 µL

Fluorescence was measured in the spectro fluorometer JASCO

Conditions:

Excitation: 460 nm
Emission spectra: 500 nm
Measurement Mode: Time course
Sensitivity: High
Temperature: 37 °C

Results: First, the Blanks are not changing significantly over time. Further results indicate a decrease of the fluorescence which increases after 30 min if ATP and Apyrase are contained in the buffer. Towards the end of the reaction the fluorescence of the RNA reduces again. If an inhibitor is added to the reaction the fluorescence stays at one level without steeper decrease.

▼2015-08-26 24. Measurement of Malachite Green Aptamer using a Fluorescent Readout

Aim: Verification of a fundamental setup (96 well/384 well plate reader) for real time monitoring of in vitro transcription using Malachite Green Aptamer. Malachite Green Aptamer will be transcribed in presence of the malachite green dye. If the monitoring works, an increase in the fluorescence should be monitored over time.

Procedure:

ssDNA (MS14 and MS15) was renatured with its complementing strand at 95 °C for 3 min

	cstock	cfinal	Volume [µL]
Transcription Buffer	10x	1x	6 µL
NTP	100 mM	4 mM	0.6 µL
DTT	1 M	1 mM	0.6 µL
Malachite green	20 mM	100 µM	0.3 µL
DNA template	10 µM	1 µM	6 µL
RiboLock	40 U/µL	0.6 U/µL	1 µL
Inorganic Pyrophosphatase	0.1 U/µL	0.002 U/µL	1 µL
T7 RNA Polymerase	1 mg/mL	0.6 mg/mL	6 µL
Millipore water			38.5 µL

Conditions:

Assay was carried out in 384 well plate for 2 hour in the TECAN
Measurement Parameters:
- Excitation: 630 nm
- Emission: 652 nm
- Excitation Bandwidth: 10 nm
- Emission Bandwidth: 10 nm
- Gain: Manual
- Gain: 60
- Flash Mode: High Sensitivity
- Integration Time: 40 µs
- Lag Time: 0 µs
- Settle Time: 0 ms
- Reading Mode: Bottom
- Kinetic Cycle
- Number of Cycles: 200
- Interval: every 30 sec

Results: The data shows an increase of fluorescence at 650 nm over time, which means that the RNA of interest was successfully transcribed.

▼2015-08-26 25. Combination of the Malachite Green Aptamer and ATP Aptamer Spinach2 to monitor ATP consumption during in vitro transcription

Aim: Real time monitoring of the decrease of ATP levels during the in vitro transcription. The ATP Aptamer Spinach2 will sense the ATP concentration in the in vitro transcription mixture. The Malachite Green Aptamer will be transcribed by the T7 RNA polymerase. We expect a decrease of the Spinach fluorescence signal and an increase of the Malachite Green Aptamer fluorescence signal over time.

Procedure:

Measure the concentration of the RNA via Nanodrop

	ATP AptamerJAWS1 Spinach2	ATP AptamerJAWS2 Spinach2
Concentration [ng/µL]	497.13	502.13

RNA was refolded in a 1x Renaturation Buffer for 3 min at 95 °C
Cool down the RNA to room temperature
For the transcription, prepare following

	cStock	cfinal	Blank w/o T7 RNA Polymerase	Functional Transcription
Transcription Buffer	10x	1x	6 µL	6 µL
NTP	100 mM	4 mM	9.6 µL	9.6 µL
DTT	1 M	10 mM	0.6 µL	0.6 µL
Malachite green	20 mM	100 µM	1 µL	1 µL
DNA template	10 µM	1 µM	6 µL	6 µL
RNA (ATP AptamerJAWS1 Spinach2 or ATP AptamerJAWS2 Spinach2)		500 nM	12 µL	12 µL
Glycerol	50 %	2.5 %	3 µL
RiboLock	40 U/µL	0.6 U/µL	1 µL	1 µL
Inorganic Pyrophosphatase	0.1 U/µL	0.002 U/µL	1 µL	1 µL
T7 RNA Polymerase	1 mg/mL	0.6 mg/mL		3 µL
Millipore water			31.8 µL	31.8 µL

Assay was performed in the TECAN Safire

Conditions:

Label 1: ATP Aptamer Spinach2

Label 2: Malachite Green Aptamer

Excitation: 460 nm

Emission: 500 nm

Excitation Bandwidth: 10 nm

Emission Bandwidth: 10 nm

Gain: Manual

Gain: 155

Flash Mode: High Sensitivity

Integration Time: 40 µs

Lag Time: 0 µs

Settle Time: 0 ms

Reading Mode: Bottom

Kinetic Cycle

Number of Cycles: 250

Interval: every 30 sec

Excitation: 630 nm

Emission: 652 nm

Excitation Bandwidth: 10 nm

Emission Bandwidth: 10 nm

Gain: Manual

Gain: 60

Flash Mode: High Sensitivity

Integration Time: 40 µs

Lag Time: 0 µs

Settle Time: 0 ms

Reading Mode: Bottom

Kinetic Cycle

Number of Cycles: 250

Interval: every 30 sec

Results: During the transcription the Blanks stayed on the same level of signal. We do not have any background in presence of the malachite green dye in the blank. Throughout the assay, no increase of fluorescence of the Malachite Green Aptamer and no decrease of the fluorescence of the ATP Aptamer Spinach2 was measured. At the beginning we can see a change in fluorescence for samples containing the ATP Aptamer Spinach2. We estimate that the ATP AptamerJAWS1 Spinach2 changes its RNA structure when it gets heated up to 37 °C. This will result in a change in fluorescence till the complete mixture has adjusted to 37 °C. In the functional assay containing all ligands, dyes and constructs, we can observe an increase of fluorescence emitted by the Malachite Green Aptamer and a decrease of the fluorescence signal given by the ATP Aptamer Spinach2/DFHBI complex that is connected to the uptake of ATP that is now bonded to the RNA strand.

▼2015-08-30 26. Analyzing the in vitro Transcription via Thin layer chromatography (TLC)

26. Analyzing the in vitro Transcription via Thin layer chromatography (TLC)

Aim: Comparison of traditional methods to analyze small molecule changes. Thin layer chromatography will be applied to analyze ATP consumption during in vitro transcription. To analyze ATP consumptions during the RNA transcription, ATP is converted to AMP after the transcription process by Apyrase

Procedure:

Transcription has been performed under the same conditions as described in previous assays such as protocol 22
As a template we used the ATP AptamerJAWS1 Spinach2
5 µL samples were taken every 5 min from the reaction
Samples were heated up to 95 °C for 10 min to inactivate the T7 RNA polymerase
After 1:30 h of incubation 0.01 U Apyrase and 4 mM CaCl2 were added to each sample and incubated for 1 h at 30 °C
2 µL of each sample was spotted on a fluorophore coated TLC Plate (ALUGRAM®Xtra SIL G/UV254, Macherey Nagel)
Mobile phase contained 4:6 1 M Ammonium acetat: ethanol
TLC ran for 4 h 45 min at room temperature

Results:

Pyrophosphate precipitate was observed during the in vitro transcription, which is a sign for a successful reaction. ATP could be converted to AMP by Apyrase as well and monitored by TLC. However TLC method is not sensitive enough to measure small changes in ATP concentration during in vitro transcription reactions.

week number 33

▼2015-07-01 11. In vitro transcription of the c-di GMP Spinach2 Variations

Aim: Synthesis of c-di GMP Aptamer Spinach2 RNA that will be used in the DFHBI Assay performed in the Tecan Safire 2

Procedure:

Follow steps as described in protocol 7 and 8 for the elution of the RNA

Results: The UV shadowing of the denaturing polyacrylamide gel electrophoresis on a fluorescent TLC shows that we were able to transcribe both RNAs.

▼2015-08-10 12. Precipitation of the c-di-GMP Mutant Spinach2 RNA

Aim: Precipitation and purification of the in vitro transcribed RNA after overnight elution (see experiment 8)

Procedure:

RNA was precipitated with 1x volume isopropanol and centrifuged at 13,000 g for 30 min, 4 °C
Precipitated RNA was washed with 70 % isopropanol and centrifuged again using the same conditions as above
The pellet was resuspended in 100 µL millipore water

Result: We were able to establish two RNA stocks of the c-di GMP Aptamer Spinach2 variations.

▼2015-08-11 13. Determination of RNA Concentration using Nanodrop

Aim: Determination of the RNA concentration which is needed for the DFHBI assay

Procedure:

1 µL of RNA stock solution was put on the Nanodrop and measured with the RNA readout program

	c-di-GMP Aptamer Spinach2	c-di-GMP Aptamer Mutant Spinach2
A260	11.303	2.874
cfinal	71 µM	18 µM

Result: Each stock solution contains enough RNA to perform the DFHBI assay.

▼2015-08-13 15. Fluorescence-spectra measurements for the Spinach2 Variations

Aim: Comparing the different Spinach2 variations in presents of DFHBI

Procedure:

the setup was prepared as described in previous Spinach2 assays like in protocol 5 or 14
tested constructs were Spinach2, c-di GMP Aptamer Spinach2 and the c-di GMP Aptamer Mutant Spinach2 and Blanks containing no RNA or no ligand
Fluorescence was measured in the spectro fluorometer JASCO

Conditions:

Excitation: 460 nm
Emission spectra: 475 nm to 600 nm
Sensitivity: High
Temperature: 37 °C

Results: Every Spinach variation except for the c-di GMP Aptamer Mutant Spinach2 showed a peak at 500 nm as expected. Furthermore we were able to detect a new peak at λ= 552 nm for the c-di GMP Aptamer Spinach2 which is higher than the peak at 500 nm.

▼2015-08-14 14. Moonlight Experiment: Test of the fluorescent readout of the c-di-GMP Spinach2 Variation

14. Moonlight Experiment: Test of the fluorescent readout of the c-di-GMP Spinach2 Variation

Aim: Testing the c-di-GMP Aptamer Spinach2 on emission in presents of the ligand c-di GMP and DFHBI and even showing the specificity of the c-di-GMP Aptamer Spinach2 in comparison to its mutant

Procedure:

Renaturing of RNA:
- RNA was renatured in the 1x Renaturing Buffer for 3 min at 95 °C
- Cool down to room temperature for 5 min
Spinach Assay:

	cStock	cfinal	Volume [µL]
HEPES KOH pH 7.5	1 M	40 mM	8
KCl	2 M	125 mM	12.5
MgCl2	1 M	3 mM	0.3
DFHBI	20 mM	100 µM	0.5
Ligand	0.01 M	100 µM	1
RNA	1 µM	0.2 µM	20
Millipore Water			57.7

Following setups were tested on fluorescence:

	Blank	Spinach2	c-di-GMP Spinach2 with water	c-di-GMP Spinach2 with ligand	c-di-GMP Mutant Spinach2 with water	c-di-GMP Mutant Spinach2 with ligand
HEPES KOH pH 7.5	8 µL	8 µL	8 µL	8 µL	8 µL	8 µL
KCl	12.5 µL	12.5 µL	12.5 µL	12.5 µL	12.5 µL	12.5 µL
MgCl2	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL	0.3 µL
DFHBI	0.5 µL	0.5 µL	0.5 µL	0.5 µL	0.5 µL	0.5 µL
Ligand				1 µL		1 µL
RNA	20 µL	20 µL	20 µL	20 µL	20 µL	20 µL
Millipore Water	58.7 µL	58.7 µL	58.7 µL	57.7 µL	58.7 µL	57.7 µL

Conditions:

Excitation: 460 nm
Emission: 500 nm
Excitation Bandwidth: 10 nm
Emission Bandwidth: 10 nm
Gain: Manual
Gain: 155
Flash Mode: High Sensitivity
Integration Time: 40 µs
Lag Time: 0 µs
Settle Time: 0 ms
Reading Mode: Bottom
Kinetic Cycle
Number of Cycles: 50
Interval: every 30 sec
Total Time: 25 min

Results: In general, the Spinach2 without ligand dependency has the highest fluorescence. The results show that the c-di-GMP Aptamer Spinach2 emits fluorescence at 500 nm. Similar data was not observed by the c-di-GMP Mutant Spinach2 which means that a mutation changes the specificity of the aptamer.

▼2015-08-14 16. Amplification of the in vitro transcription DNA template ATP Aptamer Spinach2 by PCR

Aim: Amplification and fill up of the ATP Aptamer Spinach2 construct for further applications

Procedure:

	cstock	cfinal	GC Buffer Setup Volume [µL]	HF Buffer Setup Volume [µL]
GC Buffer	5x	1x	20
HF Buffer	5x	1x		20
DMSO	100 %	5 %	5	5
dNTP			1	1
MS01 (Spinach2 forward Primer)	100 µM	1 µM	0.5	0.5
MS11 (ATP Aptamer Spinach2 reverse Primer)	100 µM	1 µM	0.5	0.5
MS08 (reverse Primer)	100 µM	2 µM	1	1
MS09 (forward Primer)	100 µM	2 µM	1	1
Phusion	2 U/ µL	0.02 U/ µL	1	1
Millipore water			70	70

Conditions:

	GC Buffer Setup	HF Buffer Setup
Denaturation Temperature	90 °C for 1 min	90 °C for 1 min
Annealing Temperature	62 °C for 30 sec	62 °C for 30 sec
Elongation Temperature	72°C for 30 sec	72°C for 30 sec
Repeat cycle 30 times
Final Elongation	72°C for 4 min	72°C for 4 min
Infinite hold at 4 °C

Analytical Agarose Gel:

2 % Agarose
70 mL TBE
2 µL Ethidium Bromide

PCR Purification:

PCR was purified by using the Quiagen PCR-Purification Kit
Purified DNA was rehydrated in 100 µL water

Result: In this set up we were able to purify the amplified DNA using the Polymerase Chain Reaction.

▼2015-08-15 17. In vitro Transcription of ATP Aptamer Spinach2

Aim: Generation of ATP Aptamer Spinach2 RNA employing the T7 RNA Polymerase

Procedure:

	cStock	cfinal	Volume [µL]
Transcription Buffer	10x	1x	10
DTT	1 M	10 mM	1
ATP	100 mM	4 mM	4
UTP	100 mM	4 mM	4
GTP	100 mM	4 mM	4
CTP	100 mM	4 mM	4
DMSO	100 %	5 %	5
DNA Template (exp. 16)			10
T7 RNA Polymerase	1 mg/mL	0.05 mg/ mL	5
Millipore Water			53

Conditions:

Incubate for 3 h, 37°C
Digest the DNA template with 10 U DNase I for 20 min
Purification of RNA by denaturing polyacrylamide gel electrophoresis (PAGE) (10% acrylamide concentration)
The gel ran for 1:30 h at 25 W
Then, the gel was eluted overnight in 15 mL 0.3 M sodium acetate pH 5.5

Results: The DNA was successfully transcribed and purified by the denaturing polyacrylamide gel electrophoresis.

▼2015-08-16 18. Precipitation of the ATP Aptamer Spinach2 RNA

Aim: Generating a stock of purified ATP Aptamer Spinach2 RNA for further applications

Procedure:

Eluate was precipitated in 1x Volume of isopropanol and centrifuged at 16,000 g, 4°C, 90 min
The RNA pellet was washed in 70 % isopropanol and centrifuged 16,000 g, 4°C, 10 min
The cleaned pellet was resuspended in 200 µL water

Result: We were able to produce a stock of purified ATP Aptamer Spinach2 RNA that we can use for the spectra measurement.

▼2015-08-16 19. Spectra measurement in the TECAN of the new ATP Aptamer Spinach2

Aim: Showing the fluorescence of the new generated ATP Aptamer Spinach2 in presence of ATP and DFHBI

Procedure:

Assay was performed under the same conditions as described in protocol 14

Results: The ATP Aptamer Spinach2 shows an increase of fluorescence in presence of ATP.

week number 32

▼2015-08-07 6. Template Preparation of the c-di-GMP Aptamer Spinach using the Polymerase Chain Reaction

Aim: Amplification of the c-di-GMP Aptamer Spinach2 and its mutant

Procedure:

	cstock	cfinal	Phusion PCR 1 Volume [µL]	Phusion PCR 2 Volume [µL]	Taq PCR Volume [µL]
Phusion Mastermix	2x	1x	25	25
*Taq* Polymerase Mastermix					25
DMSO	100 %	5 %			5
Template (gBlock: MS07 or MS10)	100 µM	1 µM	0.5	0.5	0.5
MS08 (reverse Primer)	100 µM	2 µM	1	1	1
MS09 (forward Primer)	100 µM	2 µM	1	1	1
Millipore water			17.5	17.5	17.5

MS07 c-di GMP Aptamer Spinach2

MS10 c-di GMP Aptamer Mutant Spinach2

Conditions:

	Phusion PCR 1	Phusion PCR 2	Taq PCR
Denaturation Temperature	90 °C for 1 min	90 °C for 1 min	90 °C for 1 min
Annealing Temperature	70 °C for 30 sec	62 °C for 30 sec	56 °C for 1 min
Elongation Temperature	72 °C for 30 sec	72 °C for 30 sec	72 °C for 30 sec
Repeat cycle 40x
Final Elongation	72 °C for 4 min	72 °C for 4 min	72 °C for 4 min
Infinite hold at 4 °C

Analytical Agarose Gel:

1 % Agarose
70 mL 1x Tris-Acetate EDTA buffer
2 µL Ethidium bromide

Conditions:

125 V for 30 min

Result: The PCR worked in presence of Taq Polymerase. Therefore the reaction for all primers was repeated with the Taq Polymerase (conditions as listed in this protocol). After repetition the PCR worked.

▼2015-08-07 7. In vitro Transcription of the c-di-GMP Aptamer Spinach2 and the c-di-GMP Mutant Aptamer Spinach2

Aim: Generating new RNA to show the functionality of a ligand dependent Spinach2

Procedure:

	cStock	Cfinal	Volume [µL]
Transcription Buffer	10x	1x	10
DTT	1 M	10 mM	1
ATP	100 mM	4 mM	4
UTP	100 mM	4 mM	4
GTP	100 mM	4 mM	4
CTP	100 mM	4 mM	4
DMSO	100 %	5 %	5
DNA Template			10
T7 RNA Polymerase	1 mg/mL	0.05 mg/ mL	5

Conditions:

Incubate for 3 hours
Digest the DNA template with 10 U DNase I for 20 min
Purification of RNA by denaturing polyacrylamide gel electrophoresis (PAGE) (10% acrylamide concentration)

Results: The RNA was successfully transcribed and could be purified using the PAGE.

▼2015-08-09 8. Elution of RNA using Sodium Acetate and Ethanol Precipitation

Aim: Generating a RNA stock for the DFHBI assay

Procedure: follow protocol 4

Result: The RNA for the c-di GMP Aptamer Mutant was purified and resuspended in 100 µL of millipore water. However, we were not able to purify the c-di GMP Aptamer Spinach2. Therefore we have to repeat all steps beginning from the PCR.

▼2015-08-09 9. Polymerase chain reaction to produce the c-di-GMP Aptamer Spinach2 and the c-di-GMP Mutant Aptamer Spinach2

Aim: Enhancing the yield of the PCR to increase the product concentration in the in vitro transcription of ci-di-GMP Aptamer Spinach2

Procedure:

	cstock	cfinal	GC Buffer Setup Volume [µL]	HF Buffer Setup Volume [µL]
GC Buffer	5x	1x	20
HF Buffer	5x	1x		20
DMSO	100 %	5 %	5	5
dNTP			1	1
gBlock MS07 (for the functional c-di GMPAptamer) or MS10 (for its Mutant)	100 µM	1 µM	0.5	0.5
MS08 (reverse Primer	100 µM	2 µM	1	1
MS09 (forward Primer)	100 µM	2 µM	1	1
Phusion	2 U/ µL	0.02 U/ µL	1	1

Conditions:

	GC Buffer Setup	HF Buffer Setup
Denaturation Temperature	90 °C for 1 min	90 °C for 1 min
Annealing Temperature	62 °C for 30 sec	62 °C for 30 sec
Elongation Temperature	72 °C for 30 sec	72 °C for 30 sec
Repeat cycle 30 times
Final Elongation	72 °C for 4 min	72 °C for 4 min
Infinite hold at 4 °C

Analytical Agarose Gel:

2 % Agarose
70 mL TBE
2 µL Ethidium Bromide

Conditions:

125 V for 30 min

Result: The DNA bands on the agarose gel are brighter under UV light than the ones of the fragments in the previous PCR.

▼2015-08-09 10. Purification of the PCR Products using a PCR Purification Kit

Aim: Purifying the PCR Product for the in vitro Transcription

Procedure:

Purification was performed with the QIAquick PCR Purification Kit by following their protocol

Result: The DNA was purified and is ready to be used in the in vitro transcription

week number 31

▼2015-07-27 4. Elution and Ethanol Precipitation of Spinach2 RNA

Aim: Purification of the Spinach2 for further experiments

Procedure:

RNA was eluted from the gel in 0.3 M sodium acetate pH 5.5
2.5x V of absolute ethanol was added
RNA was incubated at -80 °C over night
Precipitate the RNA at 16,000 g, 4 °C, 30 min
Wash the RNA pellet in 70 % absolute ethanol and precipitate at 16,000 g (repeat three times)
Resolve RNA in 100 µL millipore water

Result: The pellet was resuspended in 100 µL millipore water which will be the stock solution for further experiments.

▼2015-07-27 5. Analysis of the fluorescence of the Aptamer “Spinach2” in presence of the DFHBI dye

Aim: Showing the functionality of the Spinach2 in presence of DFHBI in 384-well plate format

Procedure:

Measure the concentration of the RNA using a Nanodrop:

	Concentration [µM]
Spinach2	29.8

Renaturing of RNA:
- Renature a 2 µM RNA stock with 1x Renaturing Buffer at 95 °C for 3 min
- Cool down to room temperature
Spinach Assay:

	cStock	cfinal	Volume [µL]
HEPES KOH pH 7.5	1 M	40 mM	8
KCl	2 M	125 mM	12.5
MgCl2	1 M	3 mM	0.3
DFHBI	20 mM	100 µM	0.5
Ligand	0.01 M	100 µM	1
RNA	1 µM	0.2 µM	20
Millipore Water			57.7

Following conditions were tested:

	Blank w/o DFHBI	Blank w/o RNA	Spinach2
HEPES KOH pH 7.5	8 µL	8 µL	8 µL
KCl	12.5 µL	12.5 µL	12.5 µL
MgCl2	0.3 µL	0.3 µL	0.3 µL
DFHBI		0.5 µL	0.5 µL
RNA	20 µL		20 µL
Millipore Water	58.7 µL	78.7 µL	58.7 µL

Conditions for the Tecan Safire 2:

Excitation: 460 nm
Emission: 500 nm
Excitation Bandwidth: 10 nm
Emission Bandwidth: 10 nm
Gain: Manual
Gain: 155
Flash Mode: High Sensitivity
Integration Time: 40 µs
Lag Time: 0 µs
Settle Time: 0 ms
Reading Mode: Bottom
Kinetic Cycle
Number of Cycles: 50
Interval: every 30 sec
Total Time: 25 min

Results: The assay was performed with two controls. One sample did not contain DFHBI, the other one did not have the Spinach2 RNA. The blank with no DFHBI shows a low background, whereas the control with DFHBI shows a big background (RFU= 841). However the emission of the Spinach2 containing DFHBI shows higher values (RFU= 3586) than the controls, concluding the functionality of the Spinach2 construct.

week number 30

▼2015-07-23 1. Template Preparation of Spinach2 by Polymerase Chain Reaction

Aim: Amplification of the Spinach2 fragment

Procedure:

	cStock	Cfinal	Volume [µL]
Phusion Flash Master Mix	2x	1x	50 µL
DMSO	100 %	5 %	5 µL
MS01 (Spinach2 Primer forward)	100 µM	1 µM	1 µL
MS02 (Spinach2 Primer reverse)	100 µM	1 µM	1 µL
MS08 (reverse Primer)	100 µM	1 µM	1 µL
MS09 (forward Primer)	100 µM	1 µM	1 µL
Millipore water			41 µL

PCR Conditions:

Cycle:

90 °C for 5:00 min
60 °C for 0:30 min
72 °C for 0:45 min
Repeat Cycle for 40 times
72 °C for 5:00 min
4 °C infinite hold

Analytical Agarose Gel:

2 % agarose
70 mL 1x Tris borate EDTA buffer
2 µL ethidium bromide

Conditions:

125 V for 30 min

Results: DNA was successfully amplified by PCR. Fragment has expected size of 117 bp.

▼2015-07-23 2. Ethanol Precipitation of DNA

Aim: Purification of DNA fragments < 100 bp applied for in vitro transcriptions

Procedure:
1.   0.1x Volume 3 M sodium acetate pH 5.5
2.   2.5x V absolute ethanol
3.   Incubate samples at -80 °C over night
4.   Precipitate the DNA at 13,000 g, 4 °C, 30 min
5.   Wash the DNA pellet in 70 % absolute ethanol and precipitate at 13,000 g (repeat twice)
6.   Resolve DNA in 20 µL millipore water

Result: We were able to purify the DNA.

▼2015-07-24 3. In vitro Transcription of the PCR products

3. In vitro Transcription of the PCR products

Aim: Transcribing the DNA into the RNA of Interest (ROI)

Procedure:

	cStock	cfinal	Volume [µL]
Transcription Buffer	10x	1x	10
DTT	1 M	10 mM	1
ATP	100 mM	4 mM	4
UTP	100 mM	4 mM	4
GTP	100 mM	4 mM	4
CTP	100 mM	4 mM	4
DMSO	100 %	5 %	5
DNA Template			10
T7 RNA Polymerase	1 mg/mL	0.05 mg/ mL	5
Millipore water			53

Conditions:

Incubate for 3 hours, 37 °C
Digest the DNA template with 10 U DNase I for 20 min
Purification of RNA by denaturing polyacrylamide gel electrophoresis (PAGE) (10% acrylamide concentration)

Results: The RNA was successfully synthesized by in vitro transcription.