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iGEM Team Heidelberg 2015

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week number 26

▼2015-06-25 PCR of the Fragments E, B-Hammerhead Ribozyme (Hammerhead ribozyme: HHR), EGS-Hammerhead Ribozyme and M1

Aim: Amplification of the DNA-primers for the in vitro-transcription

 

Procedure:

1. Make a 1:10 thinner of each primer by using each 9 µL water and 1 µL primer

 

	Fragment primer	Forward Primer	Reverse Primer	Lenght with out T7 promotor [bp]
Fragment E	FS01, FS02	FS 13	FS 14	66
Fragment B	FS04, FS05	FS 13	FS 15	54
Fragment EGS	FS10, FS	FS 13	FS 16	65
Fragment M1	genomic DNA	FS 08	FS 09

 

1. Fill up the following ingredients in a PCR-tube and mix them well:

   1. 1 µL of 1 µM fragment  

   2. 19 µL of water

   3. 2,5 µL of 10 µM reverse primer

   4. 2,5 µL of 10 µM foward primer

   5. 25 µL of 2x PCR Mastermix Phuion Flash

1. Put the samples into a PCR-cycler (cycle is discribed below):

   1. 98 °C for 2:30 min (denaturation)

   2. 98 °C for 0:15 min

   3. 55 °C for 0:15 min (annealing)

   4. 72 °C for 0:20 min (polymerisation)

   5. Go to step 2 and repeat for 39x

   6. 72 °C for 2:00 min

   7. 4 °C for cool down

Time total: 1 h 20 min 54 sec

1. Analyze the PCR-products by using an 1 % agarose gel with ethidumbromid:

   1. 1 % agarose

   2. 1 x TAE

   3. 20 µL/L Ethidumbromid for gel

2. Put 5 µL of each PCR-product which was mixed with 2 µL Loading Dye in a pocket

3. Fill 5 µL of a 2 log Ladder in the last pocket

4. Let the gel run for 30 min at 120 V.

5. Analyze the gel by using UV-light.

 

Conclusion:

• The PCR has worked, therefore the fragments are amplified

• Furthermore the primers (forward and reverse) were designed right since there is no visible ladder for them

▼2015-06-26 Precipitation of the PCR-DNA

Aim: Cleaning the DNA as a prepartional step for the in vitro transcripton

 

Procedure:

1. Mix each PRC-Product with 0.1x V of a 3 M NaAc and 2.5x V of absolute ethanol

2. Vortex the mixture well

3. Put the samples in the freezer for overnight

4. On the next day, spin the DNA-Ethanol mixture at 4 °C with 13000 rpm for 30 min in a centrifuge

5. Remove the ethanol from the pellet and rehydrate the pellet with 1 ml of 70 % absolute ethanol.

6. Vortex again.

7. Repeat the steps 4 to 6 for one more time and for the last cylce just 4

8. Remove the ethanol from the pellet and rehydrate the pellet with 100 µL millipore water.

9. Vortex the sample again.

▼2015-06-27 in vitro-transcription of the fragments E, B-HHR, EGS-HHR and M1 out of the DNA that was generated out of the PCR form the 25th June 2015

Aim: transcribe the PCR-samples into RNA to have the single components for the construct

Procedure for 100 µL transcription:

1. Mix together

1. 1. 4 µL of 100 mM each NTP (ATP, GTP, CTP and UTP)

   2. 10 µL of 10 x transcription buffer

   3. 1 µL of 1 M DTT

   4. 5 % of DMSO

   5. 5 µL of T7 ( 2 mg/mL)

   6. 10 µL of the DNA-template

   7. and 53 µL millipore water

2. Use a pipett to mix it up and down (do not vortex)

3. Put the samples into a heatblock at 37 °C for 3 hours

4. Add  to the mixture 5 µL of T7 polymerase and 2 µL of 1 M MgCl2 addtionally after 2 hours

5. Add 4 µL DNase I to the mixture and let it incubate at 37 °C for 20 min.

▼2015-06-27 Quantative Gel for RNA-Extraction

Aim: Purifying the RNA that was transcribed in the previous in vitro assay

Procedure:

1. Make a 8 M urea polyacrylamid gel with  Roth© mastermix, 400 µL APS and 40 µL TEMED.

2. Mix the sample with 2x Loading Dye to get a 1x Loading Dye concentration

3. Clean the pockets with TBA buffer

4. Load the entire in vitro-Transcript with Loading Dye in a pocket

5. Fill TBA buffer in all reservoirs of the gel system

6. Run the gel at 300 V for 3 hours

7. Analyze the gel through UV-Shadowing by using a TLC-plate underneath your gel

 

week number 27

▼2015-07-02 Producing fragment A by using M1 cleavage

Aim: Making a proof of principle that the M1 can cleave fragments with an external guiding strand

Procedure:

1. Measuring the transcribed RNA by using a NanoVue at 260 nm

2. Mix 5 µL of 5x buffer for fragment A, 9 µl of a 7.13 µM solution of E (RNA-Stock) and 11 µL of a 5,7 µL EGS-RNA solution

3. Mix 5 µL of 5x buffer, 17 µL of 1.87 µM M1-RNA solution and 3 µL millipore water

4. Heat up each of the mixtures to 95 °C for 5 min

5. Mix the

6. Incubate the mixture at 37 °C for 2 hours

7. Put the sample, E and EGS on a gel, therefore use the procedure discribed on 27th June 2015.

Conclusion:

• Making A did not work because we should be able to see three different. According to the results from the UV-shadowing there is just one line.

Next step:

• Optimize the assay by heating up the single fragments, thus the fragments can reach the correct secondary structure.

week number 28

▼2015-07-12 Hammerhead cleavage No. 1

Aim: Creating a functional external guiding strand and a functional B without their hammerheads

Procedure for 100 µL transcription:

1. Mix together

   1. 4 µL of 100 mM each NTP (ATP, GTP, CTP and UTP)

   2. 10 µL of 10 x transcription buffer

   3. 1 µL of 1 M DTT

   4. 5 % of DMSO

   5. 5 µL of T7 ( 2 mg/mL)

   6. 10 µL of the DNA-template

   7. and 53 µL millipore water

2. Use a pipett to mix it up and down (do not vortex)

3. Put the samples into a heatblock at 37 °C for 3 hours

4. Add  to the mixture 5 µL of T7 polymerase and 2 µL of 1 M MgCl2 addtionally after 2 hours

5. Add 4 µL DNase I to the mixture and let it incubate at 37 °C for 20 min.

6. Increase the tempeature to 95 °C and heat the samples up to 95 °C

7. Once it has hit 95 °C take the sample out and let them cool down to room temperature for 1 hour

 

In the meanwhile one can prepare the analytical gel:

1. Make a 8 M urea  20 % polyacrylamid gel  with  Roth© mastermix, 400 µL APS and 40 µL TEMED.

2. Mix the sample with 2x Loading Dye to equal a conentration of 1x Loading Dye

3. Clean the pockets with TBA buffer

4. Load the entire in vitro-Transcript with Loading Dye in a pocket

5. Fill TBA buffer in all reservoirs of the gel system

6. Run the gel at 20 W for 4 hours

7. Analyze the gel through UV-Shadowing by using a TLC-plate underneath your gel

Result:

The cleavage of B is successfully performed. EGS did not work. Therfore we will try it again. This time with two heatshocks.

week number 29

▼2015-07-15 Hammerhead cleavage of the transcripts from the 12th July 2015: No. 2

Aim: Showing that the EGS can cleave

Procedure:

1. Use the transcribed RNA from the 12th of July and heat up it up from 18 °C to 95 °C

2. Cool them down on ice for 1 hour

3. Put them back into a Thermoblock and heat it up form 10 °C to 95 °C

4. Cool them down on ice for 1 hour

 

In the meanwhile one can prepare the analytical gel:

1. Make a 8 M urea 20% polyacrylamid gel  with  Roth© mastermix, 400 µL APS and 40 µL TEMED.

2. Mix the sample with 10 µL 2x Loading Dye to get a 1x Loading Dye concentration

3. Clean the pockets with TBA buffer

4. Load the entire in vitro-Transcript with Loading Dye in a pocket

5. Fill TBA buffer in all reservoirs of the gel system

6. Run the gel at 20 W for 4 hours

7. Analyze the gel through UV-Shadowing by using a TLC-plate underneath your gel

Results:

For the second time the cleavage of the EGS did not work causing us to design a new external guiding strand that is able to cleave its hammerhead ribozyme.

week number 30

▼2015-07-23 PCR of the Fragments that are necessary for the Spinach II: proof of principle fragments

Aim: Amplification of the DNA-Spinach-primers for the in vitro-transcription

PCR III (Adavanced Style)/ Procedure (100 µL PCR):

 

	Fragments	Forward Primer	Reverse Primer	Lenght with T7 promotor [bp]
Spinach II	MS01, MS02	MS08	MS09	95
Spinach II with Theophylline Aptamer	MS01, MS05	MS08	MS09	82
Spinach II with Theophyliine Aptamer Mutant	MS01, MS06	MS08	MS09	82

 

1. Fill up the following ingredients in a PCR-tube and mix them well:

1. 1. 41 µL of water

   2. 50 µL of 2x PCR Mastermix Phuion Flash

   3. 1 µL of 100 µM foward primer

   4. 1 µL of 100 µM reverse primer

   5. 1 µL of 100 µM fragment

   6. 5 µL of DMSO

2. Put the samples into a PCR-cycler (cycle is discribed below):

   1. 95 °C for 4 min (denaturation)

   2. 95 °C for 0:30 min

   3. 62 °C for 0:30 min (annealing)

   4. 72 °C for 1 min (polymerisation)

   5. Go to step 2 and repeat for 39x

   6. 72 °C for 5:00 min

   7. 4 °C for cool down

1. Analyze the PCR-products by using an 1 % agarose gel with ethidumbromid:

1. 1. 1 % agarose

   2. 1 x TAE

   3. 20 µL/L Syber green

2. Put 5 µL of each PCR-product which was mixed with 2 µL Loading Dye in a pocket

3. Fill 5 µL of a 2 log Ladder in the last pocket

Let the gel run for 30 min at 120 V.

Analyze the gel by using UV-light.

 

Conclusion:

The gel shows that we were able to amplify the primers and to receive DNA-fragments for the spinach variations.

▼2015-07-24 Extension PCR to obtain dsDNA of Substrates for 7-18 and 10-23 DNAzyme for in vitro transcription

For the DNAzymes 7-18 and 10-23 the overlapping oligos were purchased at Sigma Aldrich, Deisenhofen, Germany. For 7-18 DNAzyme FS022 5' GGGCTAATACGACTCACTATAGGTTTTTTTTTGACTGCCGTAGGTTGCCCG 3' and FS023 5' CGGCCGCTGCTCTACCGACTGAGCTATCCGGGCGGGCAACCTACGGCAGTCAAAAAAAAACC 3' and for 10-23 DNAzyme FS028 5' GGGCTAATACGACTCACTATAGGTTTTTTTTTGACTGCCGTAGGTTGCCCG 3' and FS029 5' CGGCCGCTGCTCTACCGACTGAGCTATCCGGGCGGGCAACCTACGGCAGTCAAAAAAAAACC 3' were anealed and extended under PCR conditions (see table).

 

	cStock	cFinal	V[µL]
Primer 1	100 mM	2 µM	2
Primer 2	100 mM	2 µM	2
DMSO		5 %	5
Phusion Flash Master mix	2 x	1 x	50
H2O		ad 100 µL	41
Final			100

PCR settings:

	Temperture [°C]	Time	Cycles
Initial denaturation	95	4 min	1
Denaturation	95	30 s	39
Annealing	62	30 s
Elongation	72	1 min
Final elongation	72	5 min	1
Hold	4	∞	1

Size of the samples were checked on a 1 % agarose in TAE gel and visualized with Ethidiumbromide (EtBr) under UV light (see gel picture).

 

Results:

DNA is used for in vitro transcription as next steps.

Comment: Both constructs are actually the same as the substrate sequence Wang and coworkers, 2002 used is the same for both DNAzymes.

week number 31

▼2015-07-27 In vitro transcription of substrate RNA

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

▼2015-07-28 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

▼2015-07-29 Modification of HRP with N3

Sequence of HRP ~44 kDa, Sigma Aldrich:

>tr|K7ZW28|K7ZW28_ARMRU Peroxidase OS=Armoracia rusticana GN=HRP_A2A PE=3 SV=1

MAVTNLSTTCDGLFIISLLVIVSSLFGTSSAQLNATFYSGTCPNASAIVRSTIQQAFQSD

TRIGASLIRLHFHDCFVNGCDASILLDDSGSIQSEKNAGPNANSARGFNVVDNIKTALEN

TCPGVVSCSDILALASEASVSLTGGPSWTVLLGRRDSLTANLAGANSAIPSPFEGLSNIT

SKFSAVGLNTNDLVALSGAHTFGRARCGVFNNRLFNFSGTGNPDPTLNSTLLSSLQQLCP

QNGSASTITNLDLSTPDAFDNNYFANLQSNNGLLQSDQELFSTTGSATIAVVTSFASNQT

LFFQAFAQSMINMGNISPLTGSNGEIRLDCKKVNGS

 

All compounds were mixed as indicated in the table. Reaction was incubated at first for a couple of hours at 4 °C and then over night at 18 °C. Afterwards modified HRP was washed by the addition of MQ water and subsequent spinning at 2095 x g through an Amicon UltraFree-MC centrifugal filter with a 10 kDa cutoff. The flow through was discarded and the steps were repeated till a total volume of 30 mL water was used for washing. The sample was concentrated to a volume of ~270 µL as last step. Then it was stored at -20 °C.

	cStock	V[µL]
HRP ~44 kDa	10 mg/mL	40
H2O		160
K2CO3	2 mg/mL	100
Cu(II)SO4	1 mg/mL	25
Imidazol-sulfonyl-azide (209,61 g/mol)	2 mg/mL	15
Final		340

Conditions as Stiin, 2008.

All compounds were mixed as indicated in the table. Reaction was incubated at first for a couple of hours at 4 °C and then over night at 18 °C. Afterwards modified HRP was washed by the addition of MQ water and subsequent spinning at 2095 x g through an Amicon UltraFree-MC centrifugal filter with a 10 kDa cutoff. The flow through was discarded and the steps were repeated till a total volume of 30 mL water was used for washing. The sample was concentrated to a volume of ~270 µL as last step. Then it was stored at -20 °C.

Results:

Modification has to be proved.

When modification is confirmed N3-HRP can be used for click chemistry reaction.

▼2015-07-30 In vitro transcription of labeling RNA

Annealing of oligos:

Two complementary primers were purchased at Sigma Aldrich:

FS021: 5‘ GGGCTAATACGACTCACTATAGGCGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTCGGGCGCCA 3’

FS024: 5’ TGGCGCCCGAACAGGGACTTGAACCCTGGACCCGCGGCCGCCTATAGTGAGTCGTATTAGCCC 3’

They were annealed by mixing equal amount of both and heating it upto 95 °C for 5 min and letting it cool to rt.

In vitro Transcription:

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

 

Outlook:

As next step sample is purified over 10 % PAGE

 

▼2015-07-30 DNazyme Activity Assay

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 95 °C for 5 min and cooled on ice
• Substrate was added and the reaction was incubated at 37 °C for 1.5 h
• Reaction was stopped by storing at -20 °C
• Samples were separated on a 20 % denaturing PAGE, samples were thawed just before loading on the gel and mixed with appropriate volume of 2x loading dye
• Gel was stained with SYBR Gold and imaged with a Typhoon Scanner, GE

Results:

Amount of RNA substrate was too low to be detected by SYBR Gold. Amount of DNAzyme was too high.

Assay will be repeated with a DNase I digestion step after the reaction, to get rid of the sDNA and get a better signal from the substrate.

▼2015-08-01 Labeling 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

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.

• Samples were analyzed on an 14 % SDS-PAGE
• Gel was scanned an Typhoon Scanner using FAM mode
• Staining with silver nitrate stain according to http://proteomics.rockefeller.edu/ms_silverStaining

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.

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

 

week number 34

▼2015-08-17 Modification of substrate RNA with biotin-NTP and labeling RNA with alkyne-NTP

Substrate RNA is labeled with biotin modified NTP using Terminale Dinucleotidyl Transferase (TdT), Thermo Fisher and labeling RNA with alkyne NTPs with Poly A Polymerase (yeast), affimetrix.

Modified NTPs, all from Jena Bioscience:

• Biotin-16-dUTP
• 5-Ethynyl-dUTP (5-EdUTP)
• C8-Alkyne-dUTP
• C8-Alkyne-dCTP

	cStock	cFinal	V[µL]
TdT buffer	10 x	1 x	2.5
Substrate RNA	37,2 µM	21 µM	14.2
Biotin-NTP	1 mM	150 µM	3.75
TdT	20,000 u/mL	80 u/mL	1
CoCl2	2.5 mM	0.25 mM	2.5
H2O		Ad 25 µL	1.05
Final			25

 

	cStock	cFinal	V[µL]
PAP (yeast)	600 u/µL	24 u/µL	1
PAP buffer	5 x	1 x	5
5-EdUTP/ C8-Alkyne-dUTP/C8-Alkyne-dCTP	10 mM	400 µM	1
Labeling RNA	51.4	15 µM	3.8
H2O		Ad 25 µL	1.05
Final			25

 

• All compounds were mixed as indicated in the tables.
• Reactions were incubated for 2 h at 37 °
• Samples were stored at -20 °C oN
• RNA was precipitated by the addition of 0.1 volume of 3 M NaAc and 2.5 volume of ice cold EtOH
• Sample was stored at -20 °C for 2 h so that the RNA can precipitate
• Afterwards precipitate was pelleted by centrifugation, supernatant was discarded and pellet was dried and dissolved in 10 µL H2O
  • Substrate RNA: A260: 6.599 à 11.54 µM
  • Labeling RNA (5-EdUTP): A260: 7.123 à 18.61 µM
  • Labeling RNA C8-Alkyne-dUTP: A260: 6.458 à 16.87 µM
  • Labeling RNA C8-Alkyne-dCTP: A260: 6.378 à 16.67 µM

 

Results and Outlook:

Success of modification is checked on PAGE via a streptavidin shift for the biotin modification and via clicking Alexa 488 azide to thte alkyne modified RNA.

▼2015-08-18 Click chemistry: click alkyne modified labeling RNA to Alexa 488 azide; copper-catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC)

To check if the alkyne modified NTPs are incorporated the reactive alkyne is clicked to an azide activated Alexa 488 azide fluorophore under copper catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC). Samples are after the reaction separated on a 20 % denaturing PAGE and visualized first in the fluorescent channel for Alexa 488 and afterwards stained with SYBR Gold to visualize the RNA. Appropriate controls were performed as well.

Reaction condition can be found in the table:

	cStock	cFinal	V[µL]
Phosphate buffer pH 7.0	100 mM	50 mM	12,5
Alexa 488 azide	10 µM	400 nM	1
Alkyne modified RNA	1 µM	200 nM	5
Cu(II)SO4	2 mM	100 µM	1.25
THPTA	5 mM	500 µM	2.5
Sodium ascorbate	10 mM	1 mM	2.5
H2O		Ad 25	0.25
Final			25

Conditions as Winz, 2012.

• All compounds were mixed in the given order
• Cu(II)SO4 and THPTA and H2O were mixed before adding to the mixture to let THPTA chelat the Cu
• Lastly sodium ascorbate was added to reduce the Cu(II) to Cu(I)
• Reaction was incubated for 2 h at 37 °C and afterwards put at -20 °C

Checking success on a 20 % denaturing PAGE

• Samples were mixed with an appropriate amount of 2 x loading dye and separated on a 20 % denaturing PAGE
• Gel was scanned with a Typhoon scanner, GE in fluorescent mode using the pre-set Alexa 488 program
• Afterwards gel was stained using SYBR gold and scanned with the Typoon in SYBR Gold mode
• Steptavidin-shift in 20 % PAGE to check the success of biotin modification 2015-08-18

Result and Outlook:

Labeling of RNA with alkyne was successful and CuCCA as well. More RNA will be labeled.

▼2015-08-18 Steptavidin-shift in 20 % PAGE to check the success of biotin modification 2015-08-18

Biotin modified RNA was incubated with equimolar amounts of streptavidin before it was mixed with appropriate amounts of 2 x loading dye and loaded onto a 20 % denaturing PAGE to let biotin and streptavidin bind.

Result and Outlook:

No shift was detected. Modification reaction is repeated with more Co2+ in the reaction buffer to increase the tailing efficiency of TdT

▼2015-08-21 KW 34 – Biotin labelled substrate RNA with terminal deoxynucleotidyl transferase

For 25µL	Cstock	Cfinal	V [µL]
TdT- Buffer	10x	1x	2.5
RNA	25.05µM	10µM	10
Biotin - NTP	1mM	200µM	5
TdT	600U/µL	24U/µL	1
CoCl2	2.5mM	0.5mM	5
MQ H2O			1.5

 

The above listed substances were all mixed together and then incubated for 2 hours at 37 °C.

▼2015-08-21 Biotin labelled substrate RNA with Poly(A)Polymerase (yeast)

For 25µL	Cstock	Cfinal	V [µL]
H2O			12.14
PAP- Buffer	5x	1x	5
Biotin - NTP	1mM	100µM	2.5
RNA	28.7µM	5µM	4.36
PAP	600U/µL	24U/µL	1

 

The substances were mixed together as listed in the table above.

Eventually, having a volume of 25µl the mixture was incubated for two hours at 37 °C.

 

The Biotin labelled RNA was purified via Ethanol precipitation. Hereby sodium acetate and ethanol were added to the reagent.

 

	Volume [µL]
NaOAc (3M, pH=5.5)	2.5
EtOH	75

 

Afterwards, the Biotin labelled RNA was stored at -20 °C.

Later, the tube was centrifuged at 14.000 rpm and 4 °C for 30 min.

The supernatant was thrown away and the pellet was dissolved in 10µl MQ H2O.

▼2015-08-23 KW 35 - Diazo transfer reaction with HRP and Imidazole-1-sulfonyl azide hydrochloride

For 340µL	Cstock	V[µL]
HRP	10mg/mL	40
MQ H2O		160
K2CO3	2mg/mL	100
Cu(II)SO4•5H2O	1mg/mL	25
Imidazole-1-sulfonyl azide hydrochloride	2mg/mL	15

 

The listed substances were added chronologically and then stored for 14h and 48h at RT.

After that the solution was transferred to a Amicon UltraFree-MC centrifugal filter with a 10kDa cut off.

For a better yield HRP was purified via TCA precipitation.

 

1. 1 volume TCA (100% (w/v) to 4 volumes protein sample
2. Incubate for 10min at 4 °C
3. Centrifuge at 14K rpm for 5min
4. Remove the supernatant
5. Let the pellet dry in a heat block for 5-10min at 95 °C

 

In spite of the precipitation the concentration was very low.

Therefore, for the next approach an Amicon UltraFree-MC centrifugal filter with a 3kDa cut off was used.

week number 35

▼2015-08-25 Click Chemistry – RNA

For 100µL	Cstock	Cfinal	V[µL]
Phosphate Buffer - pH 7, 0.1M	100mM	50mM	50
Alexa 488 azide	10µM	400nM	4
RNA	1µM	200nM	20
CuSO4	50mM	100µM	0.2
THPTA	50mM	500µM	1
NaAsc	500mM	1mM	0.2
H2O			24.6

 

As the first click chemistry attempt showed positive results for one RNA sample, the reaction was repeated. All substances were mixed together as listed in the table above.

▼2015-08-25 Primer extension for substrate RNA with AU Linker

Oligos xxfs040xx and xxfs041xx (for Substrate RNA with AU linker) were annealed and extended using Phusion Flash:

	cStock	cFinal	V[µL]
Primer 1	100 mM	2 µM	2
Primer 2	100 mM	2 µM	2
DMSO		5 %	5
Phusion Flash Master mix	2 x	1 x	50
H2O		ad 100 µL	41
Final			100

PCR settings:

	Temperture [°C]	Time	Cycles
Initial denaturation	95	3 min	1
Denaturation	95	30 s	5
Annealing	62	30 s
Elongation	72	30 min
Final elongation	72	2 min	1
Hold	12	∞	1

Size of the samples were checked on a 1 % agarose in TAE gel and visualized with Ethidiumbromide (EtBr) under UV light.

▼2015-08-27 click chemistry - modified

As there were no positive results, the experiment was repeated, however, with altered concentrations:

 

For 100µL	Cstock	Cfinal	V[µL]
Phosphate Buffer - pH 7, 0.1M	100mM	50mM	50
Alexa 488 azide	10µM	400nM	4
RNA	1µL	200nM	20
CuSO4	50mM	100µM	1
THPTA	50mM	500µM	5
NaAsc	500mM	1mM	0.2
H2O			24.6

▼2015-08-28 Diazo transfer – HRP

For 340µL	Cstock	V[µL]
HRP	10mg/mL	40 and 100
MQ H2O		160 and 100
K2CO3	2mg/mL	100
Cu(II)SO4•5H2O	1mg/mL	25
Imidazole-1-sulfonyl azide hydrochloride	2mg/mL	15

 

Two samples were made with either 40µL HRP and 160µL MQ H2O or 100µL HRP and 100µL MQ H2O.

For a higher concentration each sample was centrifuged at 14k rpm for 10-15min with an Amicon UltraFree- MC centrifugal filter mixed with in total 2-3ml MQ H2O.

▼2015-08-28 Click chemistry – HRP (28-08-2015)

For 100µL	Cstock	Cfinal	V[µL]
Phosphate Buffer - pH 7, 0.1M	100mM	50mM	50
Alexa 488 azide	10µM	40µL: 2346,59nM 100µL: 465,91nM	40µL: 23,47 100µL: 4,66
Protein	40µL: 9386,36nM 100µL: 1863,63nM	40µL: 469,318nM 100µL: 93,1818nM	5
CuSO4	50mM	100µM	1
THPTA	50mM	500µM	5
NaAsc	10mM	1mM	10
H2O			40µL: 5,53 100µL: 24,34

 

The samples were incubated for 2 hours at 37 °C.

Afterwards they were analysed via SDS PAGE.

▼2015-08-30 Annealing of Primers to dsDNA for the ivT of labeling RNA

Equal amounts of Primers xxfs021xx and xxfs024xx were mixed and heated up 95 °C for 5 min. Sample was allowed to cool to RT.

Outlook:

dsDNA is used for ivT

week number 36

▼2015-08-31 Labeling of RNA with alkyne-NTP

The labeling RNA was modified with alkyne NTPs by Terminale Dinucleotidyl Transferase (TdT) and as a second approach, by Poly A Polymerase (yeast), affimetrix.

The modified NTPs are from Jena Bioscience:

• 5-Ethynyl-dUTP (5-EdUTP)
• C8-Alkyne-dUTP
• C8-Alkyne-dCTP

	cStock	cFinal	V[µL]
TdT buffer	10 x	1 x	2.5
Labeling RNA	84,97µM	15 µM	4.41
Alkyne-NTP	10 mM	400 µM	1
TdT	20,000 u/mL	80 u/mL	1
CoCl2	2.5 mM	0.25 mM	2.5
H2O		Add 25 µL	13.59
Final			25

 

	cStock	cFinal	V[µL]
PAP (yeast)	600 u/µL	24 u/µL	1
PAP buffer	5 x	1 x	5
5-EdUTP/ C8-Alkyne-dUTP/C8-Alkyne-dCTP	10 mM	400 µM	1
Labeling RNA	84.97µM	15µM	4.41
H2O		Add 25 µL	13.59
Final			25

 

• All compounds were mixed as indicated in the tables.
• Reactions were incubated for 2 hours at 37 °C
• Samples were stored at -20 °C oN

▼2015-09-01 Biotin modification of substrate RNA

For 25µL	Cstock	Cfinal	V [µL]
TdT- Buffer	10x	1x	2.5
RNA	71.804µM	10µM	3.48
Biotin - NTP	1mM	200µM	5
TdT	600U/µL	24U/µL	1
CoCl2	2.5mM	0.5mM	5
MQ H2O			8.02

 

All samples were mixed together and incubated for 2 hours at 37 °C.

▼2015-09-01 Click reaction (modified)

• new Alexa 488 with DMSO

 

For 50µL	Cstock	Cfinal	V[µL]
Phosphate Buffer - pH 7, 0.1M	100mM	50mM	25
Alexa 488 azide	10µM	400nM	2
RNA	1µM	200nM	10
CuSO4	50mM	1mM	1
THPTA	50mM	5mM	5
NaAsc	100mM	1mM	0.5
H2O			6.5

 

All samples were mixed together and then incubated for 2hours at 37 °C.

▼2015-09-02 In vitro transcription of Labeling RNA AU + substrate HRP

	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
FS_53/54 (Label AU)			20
T7 RNA Polymerase	2 mg/mL	0,1 mg/mL	5
H2O		ad 100 µL	106
Final			400

 

	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
FS_50/51 (Substrate HRP)			20
T7 RNA Polymerase	2 mg/mL	0,1 mg/mL	5
H2O		ad 100 µL	106
Final			200

 

All compounds were mixed together and incubated for 3 hours at 37 °C. Afterwards, 2µL DNAseI was added for 30min at 37 °C.

week number 37

▼2015-09-10 DNAzyme Activity: DNAzyme with ATP aptamer and calculated Kanamycin aptamer

Samples:

• 10-23 DNAzyme: xxfs032xx
• 7-18 DNAzyme: xxfs033xx
• 10-23 DNAzyme with ATP aptamer with linker: xxfs019xx
• 7-18 DNAzyme with ATP aptamer with linker: xxfs027xx
• 10-23 DNAzyme with ATP aptamer A: xxfs017xx, B: xxfs018xx
• 7-18 DNAzyme with ATP aptamer A: xxfs025, B: xxfs026xx
• 10-23 DNAzyme with calculated Kan aptamer: xxfs034xx
• 7-18 DNAzyme with calculated Kan aptamer candidate I: xxfs035xx
• 7-18 DNAzyme with calculated Kan aptamer candidate II: xxfs036xx
• 7-18 DNAzyme with calculated Kan aptamer candidate III: xxfs037xx

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 %
Adenosine in H2O:DMSO 1:2	33 mM	5 mM
H2O		ad 25 µL

 

Pipetting scheme:

#			Tris HCl ph 7.5	DNAzyme A	DNAzyme B	Substrate	NaCl	MgCl2	SDS	Adenosine	H2O	Final
1	FS032	10-23D	1,25	1,25	0,00	5,00	2,50	0,50	1,25	3,75	9,50	25,00
2	FS033	7-18D	1,25	1,25	0,00	5,00	2,50	0,50	1,25	3,75	9,50	25,00
3	FS019	10-23DmLink	1,25	1,25	0,00	5,00	2,50	0,50	1,25	3,75	9,50	25,00
4	FS027	7-18DmLink	1,25	1,25	0,00	5,00	2,50	0,50	1,25	3,75	9,50	25,00
5	FS017+18	10-23D_A+B	1,25	1,25	6,25	5,00	2,50	0,50	1,25	3,75	3,25	25,00
6	FS025+26	7-18D_A+B	1,25	1,25	6,25	5,00	2,50	0,50	1,25	3,75	3,25	25,00
7	FS032	10-23D	1,25	1,25	0,00	5,00	2,50	0,50	1,25	0,00	13,25	25,00
8	FS033	7-18D	1,25	1,25	0,00	5,00	2,50	0,50	1,25	0,00	13,25	25,00
9	FS019	10-23DmLink	1,25	1,25	0,00	5,00	2,50	0,50	1,25	0,00	13,25	25,00
10	FS027	7-18DmLink	1,25	1,25	0,00	5,00	2,50	0,50	1,25	0,00	13,25	25,00
11	FS017+18	10-23D_A+B	1,25	1,25	6,25	5,00	2,50	0,50	1,25	0,00	7,00	25,00
12	FS025+26	7-18D_A+B	1,25	1,25	6,25	5,00	2,50	0,50	1,25	0,00	7,00	25,00
13	FS032	-10-23D	1,25	1,25	0	0	2,5	0,5	1,25	0	18,25	25,00
14	FS033	-7-18D	1,25	1,25	0	0	2,5	0,5	1,25	0	18,25	25,00
15	FS019	-10-23DmLink	1,25	1,25	0	0	2,5	0,5	1,25	0	18,25	25,00
16	FS027	-7-18DmLink	1,25	1,25	0	0	2,5	0,5	1,25	0	18,25	25,00
17	FS017+18	-10-23D_A+B	1,25	1,25	6,25	0	2,5	0,5	1,25	0	12	25,00
18	FS025+26	-7-18D_A+B	1,25	1,25	6,25	0	2,5	0,5	1,25	0	12	25,00
19	Adenosine	Substrate only	1,25	0	0	5	2,5	0,5	1,25	3,75	10,75	25,00

 

 

#			Tris HCl ph 7.5	DNAzyme A	DNAzyme B	Substrate	NaCl	MgCl2	SDS	Kan	H2O	Final
20	FS032	10-23D	1,25	1,25	0	5	2,5	0,5	1,25	1,25	12	25
21	FS033	7-18D	1,25	1,25	0	5	2,5	0,5	1,25	1,25	12	25
22	FS034	Kan	1,25	1,25	0	5	2,5	0,5	1,25	1,25	12	25
23	FS035	Kan I	1,25	1,25	0	5	2,5	0,5	1,25	1,25	12	25
24	FS036	Kan II	1,25	1,25	0	5	2,5	0,5	1,25	1,25	12	25
25	FS037	Kan III	1,25	1,25	0	5	2,5	0,5	1,25	1,25	12	25
26	FS032	10-23D	1,25	1,25	0	5	2,5	0,5	1,25	0	13,25	25
27	FS033	7-18D	1,25	1,25	0	5	2,5	0,5	1,25	0	13,25	25
28	FS034	Kan	1,25	1,25	0	5	2,5	0,5	1,25	0	13,25	25
29	FS035	Kan I	1,25	1,25	0	5	2,5	0,5	1,25	0	13,25	25
30	FS036	Kan II	1,25	1,25	0	5	2,5	0,5	1,25	0	13,25	25
31	FS037	Kan III	1,25	1,25	0	5	2,5	0,5	1,25	0	13,25	25
32	Kan	Substrate only	1,25	0	0	5	2,5	0,5	1,25	1,25	13,25	25,00
33		Substrate only	1,25	0	0	5	2,5	0,5	1,25	0	14,5	25,00

 

Results and Outlook:

Positive controls worked, Adenosine dependency could be detected for one candidate.

▼2015-09-10 Click reaction of Label-, Label AU RNA [1, 2, 3]

For 51.5µL	Cstock	Cfinal	V[µL]
Phosphate Buffer - pH 7, 0.1M	100mM	50mM	25
Alexa 488 azide	10µM	400nM	2
RNA	1µM	200nM	10
CuSO4	20mM	1mM	2.5
THPTA	50mM	5mM	5
NaAsc	100mM	1mM	0.5
H2O			6.5

 

• Alexa 488 azide was solved in DMSO
• Incubation at 37 °C for 12-14 hours (overnight)