Biomod/2012/UT/Nanowranglers/Methods: Difference between revisions
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{{Template:Biomod/2012/UT/Nanowranglers}} | {{Template:Biomod/2012/UT/Nanowranglers}} | ||
=Computational methods= | |||
#'''Sequence Design''' | |||
#:Sequences of the DNA strands were designed using the program [[CircDesigNA]] | |||
#::(http://cssb.utexas.edu/circdesigna) | |||
#:Helical structures of DNA were generated by '''GIDEON''', a program for designing and analyzing complex DNA structures [4]. | |||
#'''Simulation''' | |||
#:Simulation of kinetics was generated using software '''KinTek''' [26] (Student Edition v3.0) | |||
#:(http://www.kintek-corp.com/KGExplorer/DownloadSoftware.php) | |||
= | =Experimental methods= | ||
#'''Synthesis and Purification of DNA''' | |||
: | #:'''Synthesis:''' | ||
: | #::Individual DNA strands were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA) | ||
#:'''Purification:''' | |||
: | #::All unmodified DNA strands, and reporter strand labeled with FAM were purified by denaturing polyacrylamide gel electrophoresis | ||
: | #::All other modified DNA strands were purified using HPLC by IDT without further treatment | ||
#::Concentrations of DNA solutions were obtained by measuring ultraviolet light absorption at 260 nm using Nanodrop | |||
#'''Making/Running 12% Denaturing Gel''' | |||
#:'''Making gel:''' | |||
:In a 50 mL conical tube, mix 30 mL of 20% Acrylamide in 1X TBE, 20 mL of 1X TBE Denaturing PAGE dilution buffer | #::In a 50 mL conical tube, mix 30 mL of 20% Acrylamide in 1X TBE, 20 mL of 1X TBE Denaturing PAGE dilution buffer | ||
:Add 500 | #::Add 500 μL of 10% APS and 50 µL of TEMED, mix thoroughly by inverting | ||
:Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify | #::Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify | ||
#:'''Running gel:''' | |||
:Prepare sample by mixing DNA sample with equal volume of 2X Denaturing Dye | #::Prepare sample by mixing DNA sample with equal volume of 2X Denaturing Dye | ||
:Denature sample by heating at 90 °C for 5 minutes | #::Denature sample by heating at 90 °C for 5 minutes | ||
:Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE | #::Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE | ||
:Clear lanes of excess urea by pipetting | #::Clear lanes of excess urea by pipetting | ||
:Pre-run gel at 450 V for 10-20 minutes | #::Pre-run gel at 450 V for 10-20 minutes | ||
:Load samples, run gel at 450 V with a fan | #::Load samples, run gel at 450 V with a fan | ||
# '''Making/Running 12% or 5% Native Gel''' | |||
#:'''Making gel:''' | |||
#::''For a 12% gel'': In a 50 mL conical tube, mix 15 mL of 40% Acyrlamide, 5 mL of 100% Glycerol, 5 mL of autoclaved 10X TBE, 25 mL of sterilized deionized water (sd H<sub>2</sub>O) | |||
:In a 50 mL conical tube, mix 15 mL of 40% Acyrlamide, 5 mL of 100% Glycerol, 5 mL of autoclaved 10X TBE, 25 mL of sterilized deionized water (sd | #::''For a 5% gel'': In a 50 mL conical tube, mix 6.25 mL of 40% Acyrlamide, 5 mL of 100% Glycerol, 5 mL of autoclaved 10X TBE, 33.75 mL of sterilized deionized water (sd H<sub>2</sub>O) | ||
: | #::Add 500 µL of 10% APS and 50 µL of TEMED, mix thoroughly by inverting | ||
#::Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify | |||
#:'''Running gel:''' | |||
#::Prepare sample by mixing DNA sample with 1/5 volume of 6X Orange Dye | |||
: | #::Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE | ||
: | #::Clear lanes of excess urea by pipetting | ||
: | #::Pre-run gel at 300 V for 10-20 minutes | ||
: | #::Load samples, run gel at 300 V with a fan | ||
#'''Visualizing/Excising Gel''' | |||
#:'''Visualizing gel:''' | |||
#::Add 5 µL of 10,000X SYBR Gold Nucleic Acid Gel Stain, 50 mL of 1X TBE to glass dish | |||
: | #::Remove gel from glass plates into dish, let incubate on rotator for 15 minutes | ||
: | #::Scan for Fluorescence Intensity of Storm Imager | ||
: | #:'''Excising gel:''' | ||
#::Print actual size image of gel | |||
: | #::Move gel on top of saran-wrapped PAGE glass plate, place over image | ||
: | #::Use new razor to cut DNA band, place into 1.7 mL tube | ||
: | #'''DNA Elution following Denaturing or Native PAGE''' | ||
#:'''Following Denaturing PAGE:''' | |||
#::Crush isolated DNA with plunger rod until gel is fine | |||
#::Suspend gel in 500 µL of 1X TBE | |||
: | #::Place tube on shaking incubator at 80 °C on high for 15 minutes | ||
: | #::Spin down at 14,000 rpm for 2 minutes, transfer supernatant to filter-based centrifuge tube | ||
: | #::Concentrate DNA by ethanol precipitating flow-through | ||
: | #:'''Following Native PAGE:''' | ||
: | #::Crush isolated DNA with plunger rod until gel is fine | ||
#::Suspend gel in 1 mL of 1X TBE | |||
: | #::Elute DNA overnight at 37 °C | ||
#::Spin down at 14,000 rpm for 2 minutes, transfer supernatant to filter-based centrifuge tube | |||
: | #::Add flow-through to concentrator filter until concentrated down to approximately 50 µL | ||
: | #'''Ethanol Precipitation''' | ||
: | #::Add 2.5 V of 100% ethanol, 0.1 V of 3 M NaAc (pH 5.2), 2 µL of glycogen to initial volume of solution | ||
#::Vortex, place in -80 °C freezer for 15 minutes | |||
#::Spin down at 13,000 rpm for 15 minutes at 4 °C | |||
: | #::Remove supernatant from pellet, wash pellet by adding 1 mL of 70% ethanol | ||
: | #::Spin down at 13,000 rpm for 5 minutes, discard supernatant | ||
: | #::Dry using speed vac for 10-20 minutes | ||
: | #::Resuspend DNA in sterilized deionized water | ||
: | #'''Forming/Checking Hairpins''' | ||
: | #::Denature strands by heating to 90 °C for 1 minute then slowly decreasing the temperature to 37 °C at 0.1 °C s<sup>-1</sup> | ||
: | #::Incubate strands at 37 °C | ||
#::Transfer 20 µL of each tube to new tubes | |||
#::Add 1/5 V of 6X Orange DNA Loading Dye to each tube | |||
: | #::Load 20 µL of each samples into 12% native gel | ||
: | #::Run gel at 300 V with a fan | ||
: | #::Visualize DNA | ||
:Add | #'''Walker System Assembly''' | ||
#:'''Substrate 1 (S1) Assembly:''' | |||
: | #::Combine hairpin 02aA, track strand 01Tb, hairpin 03bB | ||
: | #::Anneal by heating system at 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min<sup>-1</sup> | ||
#:'''Walker (W) Assembly:''' | |||
#::Combine W1-BHQ1, W2-BHQ1 | |||
#::Anneal by heating mixture to 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min<sup>-1</sup> | |||
: | #:'''S1-W Assembly:''' | ||
: | #::Incubate Substrate 1 (S1) and Walker (W) at 37 °C for 3 hours | ||
#:'''Substrate 2 Assembly:''' | |||
: | #::Combine hairpin 04cA, track strand 01Ta, hairpin 05dB-JOE, hairpin 06eA-TMR, 07f-FAM | ||
: | #::Anneal by heating system to 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min<sup>-1</sup> | ||
#:'''Final System (S1-W-S2) Assembly:''' | |||
:Incubate | #::Incubate S2 and S1-W at 37 °C for 3 hours | ||
: | #'''Real-time fluorescence measurement''' | ||
: | #:'''CHA Experiment:''' | ||
: | #::Prepare stock solution of RepF:RepQ complex (1:2) by annealing 20 μM RepF, 20 μM RepQ at a 1:2 volume ratio in 1XTMgK buffer | ||
#::Fold F1, F2 separately in 1XTMgK buffer by heating to 90 °C for 1 min then slowly decreasing the temperature to 37 °C at a rate of 0.1 °C s<sup>-1</sup> | |||
: | #::Prepare all samples in 1XTMgK buffer, pre-warm to 37 °C for 15–30 min before mixing | ||
#::Prevent loss due to adsorption to plastic by supplementing with 10 μM (dT<sup>21</sup>) | |||
#::Start reaction by adding F1 | |||
#::Use multichannel pipet to transfer 17 μL of reaction mixtures to a 384-well plate | |||
#::Repeat | |||
: | #::Immediately transfer plate to TECAN Safire plate reader for fluorescence measurements | ||
: | #::Set excitation, emission wavelengths to 495 nm (5-nm bandwith), 520 nm (5-nm bandwith), respectively. | ||
#::All kinetic measurements are carried out at 37 °C | |||
: | #:'''Autonomous Walker Experiments:''' | ||
#::Assemble walker using method 8 and with 100 nM track and 85 nM walker | |||
: | #::Use TECAN Safire plate reader to measure fluorescence by setting excitation and emission wavelengths to specified values, with 5-nm bandwidths | ||
: | #:::''For FAM:'' 495 nm and 520 nm | ||
: | #:::''For JOE:'' 529 nm and 555 nm | ||
< | #:::''For TAMRA:'' 559 nm and 583 nm | ||
: | #::Measure with temperature controller set to 37 °C | ||
#'''Buffers Used''' | |||
#:'''10X TBE:''' | |||
#::Tris base (pH 8.0, F.W. 121.1): 107.8 g (890 mM) <br>EDTA (F.W. 372.2): 7.44 g (20 mM) <br>Boric acid (F.W. 61.83): 55.03 g (890 mM) <br>Adjust to 1 L with sd H<sub>2</sub>O | |||
#::Filter with .0.2 μM filter | |||
#:'''10X TMgK:''' | |||
#::Tris (pH 8.0): 9.86 g Tris-HCl (F.W. 157.6) + 4.532 g Tris base (F.W. 121.1), adjust to 100 mL with sd H<sub>2</sub>O (1 M) <br>MgCl2 6H<sub>2</sub>O (F.W. 203.3): 20.33 g, adjust to 100 mL with sd H<sub>2</sub>O (1 M) <br>KCl (F.W. 74.55): 7.455 g, adjust to 100 mL with sd H<sub>2</sub>O (1 M) | |||
#::''Using solutions:'' | |||
#::Tris (pH 8.0, 1 M): 10 mL (100 mM) <br>MgCl2 (1 M): 4 mL (40 mM) <br>KCl (1 M): 15 mL (150 mM) <br>sd H<sub>2</sub>O: to 100 mL <br>Filter with .2 uM fliter |
Latest revision as of 20:56, 27 October 2012
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Computational methods
- Sequence Design
- Sequences of the DNA strands were designed using the program CircDesigNA
- Helical structures of DNA were generated by GIDEON, a program for designing and analyzing complex DNA structures [4].
- Simulation
- Simulation of kinetics was generated using software KinTek [26] (Student Edition v3.0)
- (http://www.kintek-corp.com/KGExplorer/DownloadSoftware.php)
Experimental methods
- Synthesis and Purification of DNA
- Synthesis:
- Individual DNA strands were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA)
- Purification:
- All unmodified DNA strands, and reporter strand labeled with FAM were purified by denaturing polyacrylamide gel electrophoresis
- All other modified DNA strands were purified using HPLC by IDT without further treatment
- Concentrations of DNA solutions were obtained by measuring ultraviolet light absorption at 260 nm using Nanodrop
- Synthesis:
- Making/Running 12% Denaturing Gel
- Making gel:
- In a 50 mL conical tube, mix 30 mL of 20% Acrylamide in 1X TBE, 20 mL of 1X TBE Denaturing PAGE dilution buffer
- Add 500 μL of 10% APS and 50 µL of TEMED, mix thoroughly by inverting
- Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify
- Running gel:
- Prepare sample by mixing DNA sample with equal volume of 2X Denaturing Dye
- Denature sample by heating at 90 °C for 5 minutes
- Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE
- Clear lanes of excess urea by pipetting
- Pre-run gel at 450 V for 10-20 minutes
- Load samples, run gel at 450 V with a fan
- Making gel:
- Making/Running 12% or 5% Native Gel
- Making gel:
- For a 12% gel: In a 50 mL conical tube, mix 15 mL of 40% Acyrlamide, 5 mL of 100% Glycerol, 5 mL of autoclaved 10X TBE, 25 mL of sterilized deionized water (sd H2O)
- For a 5% gel: In a 50 mL conical tube, mix 6.25 mL of 40% Acyrlamide, 5 mL of 100% Glycerol, 5 mL of autoclaved 10X TBE, 33.75 mL of sterilized deionized water (sd H2O)
- Add 500 µL of 10% APS and 50 µL of TEMED, mix thoroughly by inverting
- Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify
- Running gel:
- Prepare sample by mixing DNA sample with 1/5 volume of 6X Orange Dye
- Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE
- Clear lanes of excess urea by pipetting
- Pre-run gel at 300 V for 10-20 minutes
- Load samples, run gel at 300 V with a fan
- Making gel:
- Visualizing/Excising Gel
- Visualizing gel:
- Add 5 µL of 10,000X SYBR Gold Nucleic Acid Gel Stain, 50 mL of 1X TBE to glass dish
- Remove gel from glass plates into dish, let incubate on rotator for 15 minutes
- Scan for Fluorescence Intensity of Storm Imager
- Excising gel:
- Print actual size image of gel
- Move gel on top of saran-wrapped PAGE glass plate, place over image
- Use new razor to cut DNA band, place into 1.7 mL tube
- Visualizing gel:
- DNA Elution following Denaturing or Native PAGE
- Following Denaturing PAGE:
- Crush isolated DNA with plunger rod until gel is fine
- Suspend gel in 500 µL of 1X TBE
- Place tube on shaking incubator at 80 °C on high for 15 minutes
- Spin down at 14,000 rpm for 2 minutes, transfer supernatant to filter-based centrifuge tube
- Concentrate DNA by ethanol precipitating flow-through
- Following Native PAGE:
- Crush isolated DNA with plunger rod until gel is fine
- Suspend gel in 1 mL of 1X TBE
- Elute DNA overnight at 37 °C
- Spin down at 14,000 rpm for 2 minutes, transfer supernatant to filter-based centrifuge tube
- Add flow-through to concentrator filter until concentrated down to approximately 50 µL
- Following Denaturing PAGE:
- Ethanol Precipitation
- Add 2.5 V of 100% ethanol, 0.1 V of 3 M NaAc (pH 5.2), 2 µL of glycogen to initial volume of solution
- Vortex, place in -80 °C freezer for 15 minutes
- Spin down at 13,000 rpm for 15 minutes at 4 °C
- Remove supernatant from pellet, wash pellet by adding 1 mL of 70% ethanol
- Spin down at 13,000 rpm for 5 minutes, discard supernatant
- Dry using speed vac for 10-20 minutes
- Resuspend DNA in sterilized deionized water
- Forming/Checking Hairpins
- Denature strands by heating to 90 °C for 1 minute then slowly decreasing the temperature to 37 °C at 0.1 °C s-1
- Incubate strands at 37 °C
- Transfer 20 µL of each tube to new tubes
- Add 1/5 V of 6X Orange DNA Loading Dye to each tube
- Load 20 µL of each samples into 12% native gel
- Run gel at 300 V with a fan
- Visualize DNA
- Walker System Assembly
- Substrate 1 (S1) Assembly:
- Combine hairpin 02aA, track strand 01Tb, hairpin 03bB
- Anneal by heating system at 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
- Walker (W) Assembly:
- Combine W1-BHQ1, W2-BHQ1
- Anneal by heating mixture to 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
- S1-W Assembly:
- Incubate Substrate 1 (S1) and Walker (W) at 37 °C for 3 hours
- Substrate 2 Assembly:
- Combine hairpin 04cA, track strand 01Ta, hairpin 05dB-JOE, hairpin 06eA-TMR, 07f-FAM
- Anneal by heating system to 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
- Final System (S1-W-S2) Assembly:
- Incubate S2 and S1-W at 37 °C for 3 hours
- Substrate 1 (S1) Assembly:
- Real-time fluorescence measurement
- CHA Experiment:
- Prepare stock solution of RepF:RepQ complex (1:2) by annealing 20 μM RepF, 20 μM RepQ at a 1:2 volume ratio in 1XTMgK buffer
- Fold F1, F2 separately in 1XTMgK buffer by heating to 90 °C for 1 min then slowly decreasing the temperature to 37 °C at a rate of 0.1 °C s-1
- Prepare all samples in 1XTMgK buffer, pre-warm to 37 °C for 15–30 min before mixing
- Prevent loss due to adsorption to plastic by supplementing with 10 μM (dT21)
- Start reaction by adding F1
- Use multichannel pipet to transfer 17 μL of reaction mixtures to a 384-well plate
- Repeat
- Immediately transfer plate to TECAN Safire plate reader for fluorescence measurements
- Set excitation, emission wavelengths to 495 nm (5-nm bandwith), 520 nm (5-nm bandwith), respectively.
- All kinetic measurements are carried out at 37 °C
- Autonomous Walker Experiments:
- Assemble walker using method 8 and with 100 nM track and 85 nM walker
- Use TECAN Safire plate reader to measure fluorescence by setting excitation and emission wavelengths to specified values, with 5-nm bandwidths
- For FAM: 495 nm and 520 nm
- For JOE: 529 nm and 555 nm
- For TAMRA: 559 nm and 583 nm
- Measure with temperature controller set to 37 °C
- CHA Experiment:
- Buffers Used
- 10X TBE:
- Tris base (pH 8.0, F.W. 121.1): 107.8 g (890 mM)
EDTA (F.W. 372.2): 7.44 g (20 mM)
Boric acid (F.W. 61.83): 55.03 g (890 mM)
Adjust to 1 L with sd H2O - Filter with .0.2 μM filter
- Tris base (pH 8.0, F.W. 121.1): 107.8 g (890 mM)
- 10X TMgK:
- Tris (pH 8.0): 9.86 g Tris-HCl (F.W. 157.6) + 4.532 g Tris base (F.W. 121.1), adjust to 100 mL with sd H2O (1 M)
MgCl2 6H2O (F.W. 203.3): 20.33 g, adjust to 100 mL with sd H2O (1 M)
KCl (F.W. 74.55): 7.455 g, adjust to 100 mL with sd H2O (1 M) - Using solutions:
- Tris (pH 8.0, 1 M): 10 mL (100 mM)
MgCl2 (1 M): 4 mL (40 mM)
KCl (1 M): 15 mL (150 mM)
sd H2O: to 100 mL
Filter with .2 uM fliter
- Tris (pH 8.0): 9.86 g Tris-HCl (F.W. 157.6) + 4.532 g Tris base (F.W. 121.1), adjust to 100 mL with sd H2O (1 M)
- 10X TBE: