Template:Heidelberg/notebook/ivt/week32

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