Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Protocols

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{{Template:DeoxyriboNucleicAwesomeHeader}}
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=Protocols=
=Protocols=
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__TOC__
 
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===Things that need to be written===
 
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*Quantitating DNA
 
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*How to run a gel
 
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*How to do AFM (when we learn this)
 
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*How to analyze fluorescence experiments (when we learn this)
 
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*How to perform gel purification
 
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*How we ran experiments...
 
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=== Gel Purification ===
+
=== Oligonucleotides===
 +
Most of the sequences were from Lulu Qian’s sequences, designed as described in [1]. Modifications were added using a computer program NUPACK, and staple sequences for the rectangular origami were taken from Rothemund’s [7]. All oligonucleotides were ordered from Integrated DNA Technologies (IDT) Inc. Most of the DNA molecules were ordered standard desalted and unpurified, and modified molecules are ordered HPLC purified. Staple strands were ordered in wet plate form at 150μM in 1x TE buffer. Dried DNA was reconstituted in an appropriate amount of ddH2O, and quantified with the biophotometer.
 +
:''More detailed article about quantification protocol : [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Protocols/Quantitation|Quantitation]]''
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* 1. Anneal the complex (Q-function)
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===Buffer conditions===
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For 300ul sample, add 50ul BB_ND dye
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1x TE/Mg2+ (TE=Tris-EDTA, Tris 40 mM, EDTA 2 mM, and Magnesium acetate, 12.5 mM, pH 8.0) buffer was used for all experiments.
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170ul of sample will be loaded per lane. Samples will take 2 lanes, and one blank lane should be placed between different samples.
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* 2.  Wash the big plates. Dry the plates for 45 minutes
 
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- Feel it with fingers/ Watch out for the water flow
 
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* 3. Make a gel. 12% ND gel for purification
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===Gel Electrophoresis/ Gel Purification===
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Lulu’s recipe for 40ml of 12% ND gel which works for one gel
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5% polyacrylamide gels of 1 mm in thickness were used. Native gel was polymerized by mixing 3.75 ml 19:1 40% polyacrylamide, 1 mL 10x TAE/Mg++, 5.25 ml ddH2O, 60 μL 10% APS, 6 μL TEMED. Native gels were run at 150V at 25 ºC. 1x TAE/Mg2+ was used as a running buffer. Gels were stained with SybrGold for 15 min and then scanned with Quantity 1 FX scanner. Some complexes were annealed using PCR machine and gel purified using bigger gel. Same recipe was used to make native gel. Glycerol was used as a loading dye for the modified strands. Under UV light, the bands were cut and DNA was obtained by diffusion in 1x TE/Mg2+  buffer for 48 hours.
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- 12ml 40% Acrylamide
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:''More detailed article about gel puriciation protocol: [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Protocols/GelPurification|Gel Purification]]''
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- 4ml 10x TAE/Mg++
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- Fill up to 40ul MQ water (add 24 ml)
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- 240ul 10% APS, slow mix
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- 24ul TEMED, slow mix
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  APS should be no more than 10 days old
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* 4. Run the gel for 150V, roughly 4.5 hours ~ 6 hours (when the dye ran off the gel) depending on the size of the sample. For example, if the sample is 122nt~176nt, run it for 6 hours. Change the buffer in every 1.5 hours.
+
===DNA origami formation===
 +
Rectangular DNA origami consists of M13 viral DNA and 202 DNA staples. Staples at the side of the origami were not included in the 202 staples to avoid the stacking problem. Sometimes, a probe at the walker start site was intentionally left out, making a hole in the middle of the origami.  M13 scaffold DNA and staple strands were mixed in 1x TE/Mg2+ buffer with molar ratio of 1:4 between the M13 and staple strands. For the speed of assembly, origami were annealed at 90nM, and diluted into different final concentration for the purpose of various experiments; 15 nM of M13 were used for fluorescence experiments, and 10 nM, 5 nM, or 1 nM for or AFM experiments. Origami was annealed by heating up to 90 ºC then cooling down to 20 ºC at 1 ºC/min using an Eppendorf PCR machine. After annealing, tracks were added to the annealed origami at 1:6 ratio of M13 to tracks. Walker goal and a walker start complex which is a preannealed complex “walker – walker inhibitor – track1 – probe for track 1” were also added to the origami at 1:1 ratio of M13 to strands. Mixed solution was incubated at 37 ºC overnight.  
 +
:''More detailed article about origami puriciation protocol: [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Protocols/Origami Purification|Origami Purification]]''
 +
===Atomic force microscopy (AFM)===
 +
5 μL of sample was deposited onto a freshly cleaved mica, and 20 μL of 1x TE/Mg2+ buffer was added. 40+ μL of 1x TE/Mg2+ buffer was added to the fluid cell and the sample was scanned in a tapping mode with DNP tips.  When streptavidin was used, 1 μL of streptavidin was added to the mica, and we waited 15 or more minutes before imaging to ensure the streptavidin and biotin could bind.  The exception to this was when we used the Walker Lock-Down Protocol.
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=== SPEX protocol ===  
+
====Walker Lock-Down Protocol====
 +
To see what this protocol was used for see [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Walker Lock-Down Protocol|the AFM experiments page]].  (When we tried this protocol, the concentration of origami was too high, so we tried using 2 μL of sample instead of 5 μL, but in the future we will dilute the sample before using it, and we will stick to using 5 μL of sample.)  The sample, buffer, and streptavidin are mixed on mica as above, but after waiting 15 minutes, we do the following:
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* 1. Turn on the lamp (back side). Wait for 30 min
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1. add 20 μL of 1x TE/Mg2+ buffer to mica, then remove as much liquid as possible (40+ μL) from the mica without touching the mica.
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Bottom big on/off button is for the machine. It is always on.
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Top small one is for the lamp. If there’s somebody who will use the SPEX tomorrow, then do not turn off the lamp after the experiment.
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* 2. Turn on the computer if it’s not on.
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* 3. Software: Instrument control center
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Default: Click OK
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* 4. Temperature set up
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Click the third square icon called “Visual setup”
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Click the middle square
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Temperature control icon: Turn on and Set to 25 degreeC
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Choose T bath (internal). NOT Probe (external).
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* 5. Wash the cuvettes.
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Be careful!
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Hold only top or bottom.
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DW (10 times) 70% ethanol (twice) DW (5 times) 70% ethanol (once)
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While you wash, you can touch the side. Hold it in a stable way.
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Let it dry (upside down)
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* 6. Dry the cuvettes using lens paper.
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* 7. Click second square icon called “real time display”.
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High voltage: HV on, S 950, T 950
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Bandpass : Set 2 for everything
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* 8. Click the last square icon called “run CWA”.
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Open a file called “ROX_CWA” from e:\SPEX DATA\biomod
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Optional mode: kinetics, Integration time: 10s
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Define: Repeat acquisition: 1 min (minimum) for total of 180 min (3 hours)
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* 9. Mixing: Hold top. 350ul of 1000ul pipette all the way in. Mix it for 30 times.
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* 10. Put the cuvettes. Logo faces out!
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* 11. Start acquisition. Run sample.
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Range of the real data should not exceed 1 million (1000,000)
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Number we see is S/R  will be (100 million)
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* 12. Add more samples in the middle of SPEX experiment
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When the status is “waiting”, click “stop”.
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Don’t click anything else.
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Add and mix. After you put the cuvettes inside the box, click “no”.
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* 13. Save the data
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When the status is “waiting”, click “stop”.
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Click “yes”
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Save data in two forms (DWA datafile, multi~ test)
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Check if the data is properly saved: Check the last data.
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* If you want to see the reference, go to options: view option and display raw R.
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===How to Open SPEX Data in MATLAB===
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2. add 40 μL of 1x TE/Mg2+ buffer to mica, then remove as much liquid as possible.  Repeat this 3 times.  (In the future we may repeat this more times to remove as much excess streptavidin as possible.)
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*Open up MATLAB and use File->Import Data
+
 
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*Navigate to the raw-text (.txt) data file and open it
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3. add 20 μL of 1x TE/Mg2+ buffer to mica.
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*The table on the right should show eight rows, where the odd ones are the times associated with the signal from the row under them. The top set is sample 1; the next is 2, the next is 3, the bottom set is sample 4. Press "next."
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*Optionally right click on "data" and rename it to whatever you want. This isn't necessary unless you're afraid of your data getting overwritten eventually when you import another set.
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4. add biotinylated† walkers at a 1-to-1 ratio to the tracks or higher (excess should not affect the results, and when implemented a 1-to-1 ratio would require a volume that is too small to be pipetted, so excess is likely necessary.)
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*Click "finish."
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*Your data should be visible in the "workspace" window with the name "data" or whatever you might have renamed it to. Right click on it -> "open selection" to view it.
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5. wait for 15+ minutes before imaging sample.
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*To plot a single sample's data, use this, where "data" is the name of your data variable:
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:<syntaxhighlight lang="matlab">plot(data(1, :), data(2, :))</syntaxhighlight>
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†We have only seen the walkers when 5'-end biotinylated walkers were used, but 3'-end biotinylated walkers were only tried at a low concentration.
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*To plot all four samples' data on the same plot, use this:
+
 
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:<syntaxhighlight lang="matlab">plot(data(1, :), data(2, :), data(3, :), data(4, :), data(5, :), data(6, :), data(7, :), data(8, :))</syntaxhighlight>
+
Based on our preliminary results using this protocol, streptavidin binds to mica, and if it is ever lifted off of the mica, it can bind to walkers on origami. Thus, we will try using the above protocol, but using 1x TE/Mg2+ with 100 μM Na+ instead to minimize the amount of streptavidin that binds to mica.
-
{{Template:DeoxyriboNucleicAwesomeFooter}}
+
 
 +
===Spectrofluorometer (SPEX)===
 +
After cuvettes are cleaned with ddH2O and 70% ethanol, 10 nM - 1 uM sample was loaded in 1.5mL 1x TE/Mg2+ buffer into each cuvette for kinetics experiment in solution. For random walking experiment on origami, 200 ul of 15 nM samples were used.  Fluorescent level of each cuvette was observed in real time.
 +
 
 +
:''More detailed article about SPEX protocol: [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Protocols/SPEX|SPEX]]''
 +
 
 +
===Normalizing SPEX Data in MATLAB===
 +
:''More detailed article: [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Protocols/MATLAB|MATLAB Analysis]]''

Current revision

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Monday, September 22, 2014

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Contents

Protocols

Oligonucleotides

Most of the sequences were from Lulu Qian’s sequences, designed as described in [1]. Modifications were added using a computer program NUPACK, and staple sequences for the rectangular origami were taken from Rothemund’s [7]. All oligonucleotides were ordered from Integrated DNA Technologies (IDT) Inc. Most of the DNA molecules were ordered standard desalted and unpurified, and modified molecules are ordered HPLC purified. Staple strands were ordered in wet plate form at 150μM in 1x TE buffer. Dried DNA was reconstituted in an appropriate amount of ddH2O, and quantified with the biophotometer.

More detailed article about quantification protocol : Quantitation

Buffer conditions

1x TE/Mg2+ (TE=Tris-EDTA, Tris 40 mM, EDTA 2 mM, and Magnesium acetate, 12.5 mM, pH 8.0) buffer was used for all experiments.


Gel Electrophoresis/ Gel Purification

5% polyacrylamide gels of 1 mm in thickness were used. Native gel was polymerized by mixing 3.75 ml 19:1 40% polyacrylamide, 1 mL 10x TAE/Mg++, 5.25 ml ddH2O, 60 μL 10% APS, 6 μL TEMED. Native gels were run at 150V at 25 ºC. 1x TAE/Mg2+ was used as a running buffer. Gels were stained with SybrGold for 15 min and then scanned with Quantity 1 FX scanner. Some complexes were annealed using PCR machine and gel purified using bigger gel. Same recipe was used to make native gel. Glycerol was used as a loading dye for the modified strands. Under UV light, the bands were cut and DNA was obtained by diffusion in 1x TE/Mg2+ buffer for 48 hours.

More detailed article about gel puriciation protocol: Gel Purification

DNA origami formation

Rectangular DNA origami consists of M13 viral DNA and 202 DNA staples. Staples at the side of the origami were not included in the 202 staples to avoid the stacking problem. Sometimes, a probe at the walker start site was intentionally left out, making a hole in the middle of the origami. M13 scaffold DNA and staple strands were mixed in 1x TE/Mg2+ buffer with molar ratio of 1:4 between the M13 and staple strands. For the speed of assembly, origami were annealed at 90nM, and diluted into different final concentration for the purpose of various experiments; 15 nM of M13 were used for fluorescence experiments, and 10 nM, 5 nM, or 1 nM for or AFM experiments. Origami was annealed by heating up to 90 ºC then cooling down to 20 ºC at 1 ºC/min using an Eppendorf PCR machine. After annealing, tracks were added to the annealed origami at 1:6 ratio of M13 to tracks. Walker goal and a walker start complex which is a preannealed complex “walker – walker inhibitor – track1 – probe for track 1” were also added to the origami at 1:1 ratio of M13 to strands. Mixed solution was incubated at 37 ºC overnight.

More detailed article about origami puriciation protocol: Origami Purification

Atomic force microscopy (AFM)

5 μL of sample was deposited onto a freshly cleaved mica, and 20 μL of 1x TE/Mg2+ buffer was added. 40+ μL of 1x TE/Mg2+ buffer was added to the fluid cell and the sample was scanned in a tapping mode with DNP tips. When streptavidin was used, 1 μL of streptavidin was added to the mica, and we waited 15 or more minutes before imaging to ensure the streptavidin and biotin could bind. The exception to this was when we used the Walker Lock-Down Protocol.

Walker Lock-Down Protocol

To see what this protocol was used for see the AFM experiments page. (When we tried this protocol, the concentration of origami was too high, so we tried using 2 μL of sample instead of 5 μL, but in the future we will dilute the sample before using it, and we will stick to using 5 μL of sample.) The sample, buffer, and streptavidin are mixed on mica as above, but after waiting 15 minutes, we do the following:

1. add 20 μL of 1x TE/Mg2+ buffer to mica, then remove as much liquid as possible (40+ μL) from the mica without touching the mica.

2. add 40 μL of 1x TE/Mg2+ buffer to mica, then remove as much liquid as possible. Repeat this 3 times. (In the future we may repeat this more times to remove as much excess streptavidin as possible.)

3. add 20 μL of 1x TE/Mg2+ buffer to mica.

4. add biotinylated† walkers at a 1-to-1 ratio to the tracks or higher (excess should not affect the results, and when implemented a 1-to-1 ratio would require a volume that is too small to be pipetted, so excess is likely necessary.)

5. wait for 15+ minutes before imaging sample.

†We have only seen the walkers when 5'-end biotinylated walkers were used, but 3'-end biotinylated walkers were only tried at a low concentration.

Based on our preliminary results using this protocol, streptavidin binds to mica, and if it is ever lifted off of the mica, it can bind to walkers on origami. Thus, we will try using the above protocol, but using 1x TE/Mg2+ with 100 μM Na+ instead to minimize the amount of streptavidin that binds to mica.

Spectrofluorometer (SPEX)

After cuvettes are cleaned with ddH2O and 70% ethanol, 10 nM - 1 uM sample was loaded in 1.5mL 1x TE/Mg2+ buffer into each cuvette for kinetics experiment in solution. For random walking experiment on origami, 200 ul of 15 nM samples were used. Fluorescent level of each cuvette was observed in real time.

More detailed article about SPEX protocol: SPEX

Normalizing SPEX Data in MATLAB

More detailed article: MATLAB Analysis
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