Biomod/2011/LMU/FolD'N'Assemble/Protocols: Difference between revisions

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{| class="wikitable" cellpadding="15"
{| class="wikitable" cellpadding="15"
|style="width:10em"| [[Biomod/2011/LMU/FolD%27N%27Assemble|<span style="color:black;">'''HOME'''</span>]]
|style="width:10em"| [[Biomod/2011/LMU/FolD%27N%27Assemble|<span style="color:black;">'''HOME'''</span>]]
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|style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Project|<span style="color:black;">'''THE PROJECT'''</span>]]
|style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Project|<span style="color:black;">'''THE PROJECT'''</span>]]
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|style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Results|<span style="color:black;">'''RESULTS'''</span>]]
|style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Methods|<span style="color:black;">'''METHODS'''</span>]]
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|style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Protocols|<span style="color:black;">'''PROTOCOLS'''</span>]]
|style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Protocols|<span style="color:black;">'''PROTOCOLS'''</span>]]
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|style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Roadmap|<span style="color:black;">'''SCHEDULE'''</span>]]
|style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Labbook|<span style="color:black;">'''LAB BOOK'''</span>]]
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|style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Results|<span style="color:black;">'''RESULTS'''</span>]]
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|style="width:10em; border-top: 1pt black solid"| '''TEAM'''
|style="width:10em; border-top: 1pt black solid"| '''TEAM'''


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[[User:Ralf Weidner|<span style="color:black">Ralf Weidner</span>]]
[[User:Ralf Weidner|<span style="color:black">Ralf Weidner</span>]]


 
[[User:Miranda Roßmann|<span style="color:black">Miranda Roßmann</span>]]


[http://www.softmatter.physik.uni-muenchen.de/tiki-index.php?page=CVschittler <span style="color:black">Verena Schüller</span>]
[http://www.softmatter.physik.uni-muenchen.de/tiki-index.php?page=CVschittler <span style="color:black">Verena Schüller</span>]
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= Design of the structure =
The structure was designed by using the CAD software "caDNAno". A very good tutorial with further information and examples is available at
http://cadnano.org/
== DNA Sequences ==
Here you can find the cadnano source file and all other DNA sequences used for the project; The design is modular and consists out of the core structure (monomer) and connection staples that are used for dimerisation
*Cadnano file of the core structure staples + dimerisation edge staples (use m13/p8634 scaffold)
http://openwetware.org/images/4/44/Nanopill.json
Note: The staples for the nanopill are color coded. Red signifies edge staples that are used for dimersation but are not important for the stability of the main structure, they can be removed or replaced with different dimerisation methods
CS - only core staples
ES - end staples(regular)
The ES are added before folding to create fixed dimers. These dimers are used for reference in gel electrophoresis
*connection strands including the i-motif
There are 5 imotif strands and 5 complimentary strands, the strands are numbered in roman numbers (I to V); the sequences Ia-Va have two more mismatches.
http://openwetware.org/wiki/Image:Connectionstrands.xls
*Strand with attached Cy5 dye, used for loading experiments
5` -> 3´
Cy5-TTT TGT TAG TGT TAG TGT TAG ACT A


= Folding of the core structure =
= Folding of the core structure =


To fold the structure you need a buffered 10:1 solution of staple and scaffold DNA; Mg2+ is added in the form of MgCl2 to reduce the repulsion of the closely packed negative DNA backbones and stabilize the structure, the optimal concentration of MgCl2 has to be determined by experimentation and depend on the structure
To fold the structure you need a buffered 10:1 solution of staple/scaffold DNA; the DNA staples can be ordered from various companies that specialize in DNA synthesis(MWG in our case), the scaffold can be prepared using the method described in the supplementary section of


(for the nanopill use 18mM MgCl2, m13/p8634 scaffold; the staple sequences can be found here[LINK])
"A primer to scaffolded DNA origami" by Castro et al. (2009)


http://www.nature.com/nmeth/journal/v8/n3/extref/nmeth.1570-S1.pdf
Mg2+ is added in the form of MgCl2 to reduce the repulsion of the closely packed negative DNA backbones and stabilize the structure, the optimal concentration of MgCl2 has to be determined by experimentation and depends on the structure
(for the nanopill use 18mM MgCl2, m13/p8634 scaffold; the staple sequences can be found here http://openwetware.org/images/4/44/Nanopill.json


== Sample preparation for folding (25 μl) ==
== Sample preparation for folding (25 μl) ==
Line 50: Line 100:
== Annealing ==
== Annealing ==


Anneal the samples in a thermocycler; in the annealing process the DNA is completely denaturized by heating and then slowly cooled, allowing for optimal strand configuration; optimal annealing times have again to be determined by experimentation and depend on the structure
Anneal the samples in a thermocycler; in the annealing process the DNA is completely denaturized by heating and then slowly cooled, allowing for optimal strand configuration; the best annealing time has again to be determined by experimentation and depends on the structure


(for the nanopill 55h hours proofed to be the most effective, acceptable results can already be achieved after 4h of annealing)
(for the nanopill 55h hours proofed to be the most effective, acceptable results can already be achieved after only 4h)


Thermocycler programs:
Used thermocycler programs:


* 4h
* 4h
Line 128: Line 178:
* once the gel is solid, fill the gel box with TBE/11mM MgCl2 buffer
* once the gel is solid, fill the gel box with TBE/11mM MgCl2 buffer
* remove comb, cool gel box with ice
* remove comb, cool gel box with ice
* pipette appropriate amount of annealed sample into gel wells, the sample should consist roughly of 1/5 loading 6x loading buffer
* pipette appropriate amount of annealed sample into gel wells, the sample should consist roughly of 1/5 6x loading buffer
* for reference a lane each with DNA Ladder and scaffold should be added to the gel for reference; the ladder allows to estimate the size of structures
* a lane each with DNA ladder and scaffold should be added to the gel for reference; the ladder allows to estimate the size of structures
* apply 70V for 3h, the current depends on salt concentration an should not exceed 200 mA
* apply 70V for 3h, the current depends on salt concentration an should not exceed 200 mA


=== Gel staining ===
=== Gel staining ===
* to make the DNA visible under UV light stain the gel in a 0.5x TBE and Etidium Bromide solution for 30 min
* to make the DNA visible under UV light stain the gel in a 0.5x TBE and etidium bromide solution for 30 min
* afterwards wash the gel 10 min in a clean 0.5x TBE solution to remove excess EtBr
* afterwards wash the gel 10 min in a clean 0.5x TBE solution to remove excess EtBr
* examine the gel in uv chamber, DNA cluster of similar size should form distinct bands
* examine the gel in uv chamber, DNA cluster of similar size should form distinct bands
Line 142: Line 192:
* fill freeze 'N squeeze filter with the gel pieces, spin at 5000 rpm for 5 min
* fill freeze 'N squeeze filter with the gel pieces, spin at 5000 rpm for 5 min
* column can be discarded, freeze sample for later use  
* column can be discarded, freeze sample for later use  
= Loading with Cy5 =
* for statistical loading use 1 μM of Cy5 strands
* for attaching the Cy5 strands to i-motif strands use the standard 10:1 ratio of staple/scaffold given above


= pH value adjustment =
= pH value adjustment =


== Buffer exchange ==
== Buffer exchange ==
Prepare the needed buffers with adjusted pH value


Use 2 tubes and one filter per sample
Use 2 tubes and one filter per sample
Line 163: Line 216:
== Buffer preparation ==
== Buffer preparation ==


* TE buffer recipe
 
* TE buffer solution


for 1 l of 10x stock solution
for 1 l of 10x stock solution


* 100 ml 1M Tris-HCl pH 7.5 or 8.0
- 100 ml 1M Tris-HCl pH 7.5 or 8.0
* 20 ml 0.5M EDTA pH 8.0
* 880 ml ddH2O


* TBE buffer solution
- 20 ml 0.5M EDTA pH 8.0
- 880 ml ddH2O


0.5xTBE Stock for 0.1 Liter (pH 8.2)


* Tris-base 5.4 g
* TBE buffer solution
* Boric Acid 2.75 g
* EDTA 20mL 0.5M (or 0.292 g)
* Add dH2O to 0.1 L
* MgCl2: 2.236 g for 11mM in 10L


* PBS buffer
for 1 liter of 5X TBE mix


1x PBS buffer solution contains
- 54 g Tris Base


* 137 mM NaCl
- 27.5 g Boric Acid
* 2.7 mM KCl
* 4.3 mM Na2HPO4
- 20 ml EDTA pH 8
* 1.47 mM KH2PO4


* Loading buffer
- fill up with dH2O


Loading buffer contains


* 6x Agarose Gel Loading buffer
* PBS buffer solution
* 30% Glycerol weight-to-volume in water
* 0.025% Xylene cyanol
* 0.025% Bromophenol blue


1x PBS buffer solution contains


* 1kb DNA Ladder
- 137 mM NaCl


1kb DNA Ladder contains
- 2.7 mM KCl


* Gel Loading Dye Blue 6x from New Englan Biolab - 100 μl
- 4.3 mM Na2HPO4
* 1kB DNA-Ladder from New England Biolabs - 100 μl
* Distilled water - 400 μl
* Total volume - 600 μl


- 1.47 mM KH2PO4




* Loading buffer


Loading buffer contains


- 6x Agarose Gel Loading buffer


- 30% Glycerol weight-to-volume in water


- 0.025% Xylene cyanol


= Lab notebook =
- 0.025% Bromophenol blue
 
 
 
===May===
====05-13-11(Timon, Alex, Ralf)====
[[Image:20110513 4h-24h-55h 10-14-18Mg.png| thumb |upright=1.5|05-13-11, Florescence image with different annealing times and MgCl2 concentrations, 2%
agarose gel, 3h running time, 70V, 0.5X TBE running buffer; From left to right: 1kb ladder, scaffold, sample 1-10]]
 
* We tried to find the optimal MgCl2 concentration and annealing time for our structure, a range of different samples were prepared for comparison. We also made a first attempt to dimerise monomers.
 
{| {{table}}
|-
! SampleNr. !! Annealing time !! MgCl(mM)!!
|-
| 1 || 4h || 10
|-
| 2 || 4h || 14
|-
| 3 || 4h || 18
|-
| 4 || 24h || 10
|-
| 5 || 24h || 14
|-
| 6 || 24h || 18
|-
| 7 || 24h || 14 ||dimers(2ES)
|-
| 8 || 55h || 10
|-
| 9 || 55h || 14
|-
| 10 || 55h || 18
|-
|}
 
* Gel electrophoresis was used to test the samples; 55h and 18 mM MgCl2 produced the best results; dimerisation worked quite well
 
* Extracted samples 1, 4 and 7 for further TEM analysis
 
 
 
 
 
 
====05-16-11 (Timon, Alex, Ralf)====
* Further experiments with MgCl2 concentration(16-18 mM), this time also with dimers.
[[Image:20110516.JPG|thumb|upright=1.5|05-16-11, Florescence image with varying MgCl2 concentrations and Dimers, 2%
agarose gel, 3h running time, 70V, 0.5X TBE running buffer;
From left to right: ladder, scaffold, sample 1-6]]
{| {{table}}
|-
! SampleNr. !! Annealing time !! MgCl(mM)!!
|-
| 1 || 55h || 16
|-
| 2 || 55h || 18
|-
| 3 || 55h || 16 ||dimers(2ES)
|-
| 4 || 55h || 18 ||dimers(2ES)
|-
| 5 || 55h || 16 ||dimers(4ES)
|-
| 6 || 55h || 18 ||dimers(4ES)
|-
|}
 
 
 
 
 
 
====05-17-11(Timon, Alex, Ralf)====
We took TEM (transmission electron microscope) images of the extracted samples.
 
[Here be TEM images]
 
 
 
 
 
====05-18-11(Timon, Alex, Ralf)====
Prepared new samples for next week, planning to do a test of structure stability in different pH environments; 200μL, 18mM MgCl, 55h
 
 
 
 
 
====05-23-11(Timon, Alex, Ralf)====
Prepared pH adjusted TE buffers for stability tests; used HCl for lowering the pH value
 
*pH 4,5
*pH 5
*pH 6
*pH 8
 
 
 
 
 
====05-24-11(Alex, Ralf, Timon)====
*sample preparation for pH stability Experiment
 
*prepared 3 samples
 
*exchanged standard buffer with pH adjusted buffer using amicon centrifugal filters
 
*~24 h exposure to pH values of 4.5, 5 and 6, 1x TE buffer, 18mM MgCl2
 
 
 
 
 
 
====05-25-11(Alex)====
*Ran the pH adjusted samples in 2% agarose gel, UV images are inconclusive, going to repeat experiment on Mo 05-30-11
 
 
 
 
 
===05-27-11(Alex)===
*prepared samples to repeat the buffer pH adjusted gel, 150 μL, standard recipe
 
 
 
 
 
===05-30-11(Alex)===
[[Image:Gel_ph_053011.png|thumb|upright=1.5|05-30-11, Florescence image with samples at different pH values, 2%
agarose gel, 3h running time, 70V, 0.5X TBE running buffer;left to right, Ladder 1kb, Scaffold, pH 8(control sample), pH 8, pH 6, pH 5, pH 4.5]]
*ran a gel with 5 samples
*pH 8 (control sample, no buffer exchange at all)
*pH 8 (no pH change but performed the buffer exchange procedure)
*pH 6
*pH 5
*pH 4.5
*origami seems stable under changing pH conditions, extracted sample with pH 5 for further TEM analysis
 
 
 
 
 
 
 
====05-31-11(Timon)====
*made new staple mix for CS(Core staples) 200nM
*started annealing new samples with i-motif
 
 
 
 
 
 
====06-03-11(Timon)====
[[Image:20110603 1.i-motif.JPG|thumb|upright=1.5|06-03-11, Florescence image with i-motif dimers, 2%
agarose gel, 3h running timeleft to right, Ladder 1kb, Scaffold, momomers, dimers(4b), dimers with i-motif]]
Ran a gel(2% agaroses) with 3 samples to test dimerisation with the i-motif connections
*monomers(cs)
*dimers(cs+4es)
*dimers + i-motif (cs+i-m)
 
 
 
 
 
 
 
====06-07-11(Timon)====
I Prepared new samples
*25μL cs
*25μL cs+4es
*25μL cs+i-motif I+II
*100μL cs+i-motif II-V
 
 
 
 
 
====06-10-11(Timon)====
[[Image:20110610 cs, cs+4es,imI+II,3ximI-V.JPG|thumb|upright=1.5|06-10-11,Florescence image with i-motif dimers, 2%
agarose gel, 3h running timeleft to right, Ladder 1kb, Scaffold, momomers, dimers(4b), dimers with i-motif I+II, 3x dimers with i-motif I-V]]
I ran a gel (2% agarose)
*cs
*cs+4es
*cs+i-motif I+II
*3x cs+i-motif I-V
 
[[Image:20110610.png|thumb|upright=1.5|06-10-11, Florescence image with i-motif dimers, 2%
agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, i-motif I-V pH 8, i-motif I-V pH 8 with buffer change, i-motif I-V pH 5 with buffer change]]
 
I cut out the three i-motifs (I-V) and did a buffer exchange with the centrifuge.
Then i made a second gel
* i-motif I-V pH 8
* i-motif I-V pH 8 with buffer change
* i-motif I-V pH 5 with buffer change
 
 
 
 
 
 
 
====06-14-11(Timon)====
Prepared new samples
*25μL i-motif I-II
*25μL i-motif I-III
*25μL i-motif I-IV
*25μL i-motif I-V
 
 
 
 
 
 
====06-17-11(Timon)====
 
[[Image:20110617 imotifconnections.JPG|thumb|upright=1|06-17-11, Florescence image with i-motif dimers, 2%
agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, i-motif I-II, i-motif I-III, i-motif I-IV, i-motif I-V]]
 
[[Image:20110617 imotifphchange.JPG|thumb|upright=1|06-17-11, Florescence image with i-motif dimers, 2%
agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, Monomers, i-motif I-V pH 5,6]]
 
2% agarose gel
*4,6,8,10 i-motif connections
* Didn't work out. Probably something wrong with the gel.
* I cut out the I-V bande(10 connections)and tried to change the i-motif by pH.
* Didn't work out either. At least the ladder worked, because i mixed it myself.
 
New samples
*75μL i-motif I-II
*75μL i-motif I-III
*75μL i-motif I-IV
*75μL i-motif I-V
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
====06-17-11(Alex)====
 
[[Image:061711-buffers_lab.gif‎|thumb|upright=1|06-17-11, Florescence image with ES dimers, 2%
agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, (1):Monomers, 1x TE, untreated, pH 8, (2):Monomers, 1x TE, buffer
exchange, pH 8 (3):Monomers, 1x TE, buffer exchange, pH 5, (4):Monomers,
PB, buffer exchange, pH 5.3 (5):Monomers, PBS, buffer exchange, pH 5.8,
(6):Dimers 2ES, 1x TE, buffer exchange, pH 8 (7):Dimers 2ES, 1x TE, buffer
exchange, pH 5,(8):Monomers, PB, buffer exchange, pH 5.3 (9):Dimers 2ES,
PBS, buffer exchange, pH 5.8]]
 
 
*Exposed monomers/dimers to different buffer solutions, to wit, phosphate buffer and PBS(Phosphate buffer saline), structures are stable in all buffers/the entire pH range
 
*Prepared some samples for a 24h exposure test
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
====06-20-11(Timon)====
[[Image:20110620 imotifconnectionsph.JPG|thumb|upright=1.5|06-20-11, Florescence image with i-motif dimers, 2%
agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, 4,6,8,10 i-motif connections TE pH8/ 4,6,8,10 i-motif connections Phosphat buffer pH 5,3/4,6,8,10 i-motif connections PBS pH 5,6]]
 
2% agarose gel
*4,6,8,10 i-motif connections TE pH8
*4,6,8,10 i-motif connections Phosphat buffer pH 5,3 (buffer change)
*4,6,8,10 i-motif connections PBS pH 5,6 (buffer change)
 
 
 
 
 
 
====06-20-11(Alex)====
 
* 24h PBS/PB exposure samples all aggregated, propably something wrong with the gel and/or folding process 
 
 
 
 
 
 
====07-08-11(Timon)====
* Prepared new samples
 
 
 
 
 
 
 
====07-11-11(Timon)====
[[Image:20110711 verbindungen ph1.JPG|thumb|upright=1.5|07-11-11, Florescence image with i-motif dimers, 2%
agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, 4,6,8,10 i-motif connections TE pH8/ 4,6,8,10 i-motif connections Phosphat buffer pH 5,3/4,6,8,10 i-motif connections PBS pH 5,6]]
* Made a buffer change with Amicon filters
* Used buffers: TE(pH 8), Phosphat buffer(pH 5,5), PBS (18mM MgCl, pH 5,6)
* Then i ran a 2% agarose gel. Containers did not fold correctly.
 
 
 
 
 
 
====07-12-11(Timon)====
* Took new scaffold from the stock and prepared new samples
 
 
 
 
 
 
====07-15-11(Timon)====
[[Image:20110715 mono dim 4-10verbindungen ph8 scalt1.JPG|thumb|upright=1.5|07-15-11, Florescence image with i-motif dimers, 2%
agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold,monomers, dimers, 4,6,8,10 i-motif connections TE pH8, old scaffold]]
2% agarose gel to see if the new scaffold from the stock works. It did not.
 
 
 
 
 
 
 
====08-19-11(Timon)====
[[Image:20110819 sc8634 phagen.JPG|thumb|upright=1.5|08-19-11, Florescence image with different scaffold solutions, 2%
agarose gel, 3h running time, left to right, Ladder 1kb,new scaffold1, new scaffold2, old scaffold, new phage, old phage]]
 
*A new scaffold stock(p8634) was produced in the lab. I ran a 2% agarose gel to compare the old and the new scaffolds and phages.
*Prepared new samples
 
 
 
 
 
 
====08-22-11(Alex)====
[[Image:082211-CS-ES-imotif.gif|thumb|upright=1.5|08-22-11, Florescence image with i-motif dimers, 2%
agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, momomers, dimers, dimers with i-motif I-II, I-III, I-IV and I-V]]
 
*Ran a comeback gel with momomers/dimers and the new scaffold, all samples fold nicely
 
*Extracted imotif samples I-II, I-IV and I-V for TEM analysis, if needed
 
*Prepared new samples(50 μl) of monomers/dimers for testing the opening mechanism on thursday
 
 
 
 
 
 
 
 
====08-25-11(Alex)====
[[Image:082511imotifopening.png|thumb|upright=1.5|08-25-11, Florescence image with i-motif dimers, 2%
agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, momomers, dimers, dimers with i-motif I-II (unmodified/pH4.5), I-III (unmodified/pH4.5), I-IV (unmodified/pH4.5) and I-V (unmodified/pH4.5)]]
 
*Tried inducing the i-motif formation by buffer exchange(1x TE pH 4.5)to seperate the dimers, but it didn't work out as intended and there appears to be no i-motif formation at all
 
*pH adjusted samples have moved faster through the gel, need to take TEM images
 




* 1kb DNA Ladder


1kb DNA Ladder contains


- Gel Loading Dye Blue 6x from New Englan Biolab - 100 μl


====09-02-11(Timon)====
- 1kB DNA-Ladder from New England Biolabs - 100 μl
We made new TE buffers:
*pH 3, 180mM MgCl
*pH 4.5, 100mM MgCl
*pH 4.5, 50mM MgCl
*pH 4.5, 500mM NaCl


*The pH value was adjusted by ading HCl
- Distilled water - 400 μl


*Prepared new samples
- Total volume - 600 μl

Latest revision as of 16:56, 2 November 2011


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HOME
THE PROJECT
METHODS
PROTOCOLS
LAB BOOK
RESULTS
TEAM

Timon Funck

Aleksej Belizki

Ralf Weidner

Miranda Roßmann

Verena Schüller

Prof. Tim Liedl


Design of the structure

The structure was designed by using the CAD software "caDNAno". A very good tutorial with further information and examples is available at

http://cadnano.org/

DNA Sequences

Here you can find the cadnano source file and all other DNA sequences used for the project; The design is modular and consists out of the core structure (monomer) and connection staples that are used for dimerisation

  • Cadnano file of the core structure staples + dimerisation edge staples (use m13/p8634 scaffold)

http://openwetware.org/images/4/44/Nanopill.json

Note: The staples for the nanopill are color coded. Red signifies edge staples that are used for dimersation but are not important for the stability of the main structure, they can be removed or replaced with different dimerisation methods

CS - only core staples

ES - end staples(regular)

The ES are added before folding to create fixed dimers. These dimers are used for reference in gel electrophoresis

  • connection strands including the i-motif

There are 5 imotif strands and 5 complimentary strands, the strands are numbered in roman numbers (I to V); the sequences Ia-Va have two more mismatches.

http://openwetware.org/wiki/Image:Connectionstrands.xls

  • Strand with attached Cy5 dye, used for loading experiments

5` -> 3´ Cy5-TTT TGT TAG TGT TAG TGT TAG ACT A

Folding of the core structure

To fold the structure you need a buffered 10:1 solution of staple/scaffold DNA; the DNA staples can be ordered from various companies that specialize in DNA synthesis(MWG in our case), the scaffold can be prepared using the method described in the supplementary section of

"A primer to scaffolded DNA origami" by Castro et al. (2009)

http://www.nature.com/nmeth/journal/v8/n3/extref/nmeth.1570-S1.pdf


Mg2+ is added in the form of MgCl2 to reduce the repulsion of the closely packed negative DNA backbones and stabilize the structure, the optimal concentration of MgCl2 has to be determined by experimentation and depends on the structure

(for the nanopill use 18mM MgCl2, m13/p8634 scaffold; the staple sequences can be found here http://openwetware.org/images/4/44/Nanopill.json

Sample preparation for folding (25 μl)

  • 2.5 μl Scaffold 100 nM
  • 2.5 μl 10x TE buffer solution + MgCl2
  • 7.5 μl Staple mix 200 nM
  • 12.5 μl ddH2O

Annealing

Anneal the samples in a thermocycler; in the annealing process the DNA is completely denaturized by heating and then slowly cooled, allowing for optimal strand configuration; the best annealing time has again to be determined by experimentation and depends on the structure

(for the nanopill 55h hours proofed to be the most effective, acceptable results can already be achieved after only 4h)

Used thermocycler programs:

  • 4h

1=80C for 5:00 min

2=79C for 4:00 min

-1C per cycle

3=Goto 2, 19 times

4=60C for 2:15 min

-0.5C per cycle

5= Goto 4, 71 times

6=End


  • 24h

1=80C for 5:00 min

2=79C for 4:00 min

-1C per cycle

3=Goto 2, 19 times

4=60C for 18:00 min

-0.5C per cycle

5= Goto 4, 71 times

6=4C 10 h

7=Goto 6, 4 times

8=End


  • 55h

1= 80C for 5:00 min

2= 79C for 4:00 min

-1C per cycle

3=Goto 2, 19 times

4=60C for 46:00 min

-0.5C per cycle

5=Goto 4, 71 times

6=End

Gel electrophoresis to evaluate the folding results

Preparation of a 2% agarose gel

  • weight 3 g agarose in a beaker
  • fill up to 150 ml with 0.5x TBE buffer
  • boil in microwave until the agarose is completely dissolved, pan beaker a couple of time, boil again
  • cool it in an bucket of ice until hand warm
  • add 1.65 ml of 1 M MgCl2 solution (gives a total MgCl2 concentration of 11mM)
  • fill the gel tray and install comb immediately
  • once the gel is solid, fill the gel box with TBE/11mM MgCl2 buffer
  • remove comb, cool gel box with ice
  • pipette appropriate amount of annealed sample into gel wells, the sample should consist roughly of 1/5 6x loading buffer
  • a lane each with DNA ladder and scaffold should be added to the gel for reference; the ladder allows to estimate the size of structures
  • apply 70V for 3h, the current depends on salt concentration an should not exceed 200 mA

Gel staining

  • to make the DNA visible under UV light stain the gel in a 0.5x TBE and etidium bromide solution for 30 min
  • afterwards wash the gel 10 min in a clean 0.5x TBE solution to remove excess EtBr
  • examine the gel in uv chamber, DNA cluster of similar size should form distinct bands

Gel Purification

  • cut out the piece of gel with the sample using a razor blade
  • cut it into small pieces
  • fill freeze 'N squeeze filter with the gel pieces, spin at 5000 rpm for 5 min
  • column can be discarded, freeze sample for later use

Loading with Cy5

  • for statistical loading use 1 μM of Cy5 strands
  • for attaching the Cy5 strands to i-motif strands use the standard 10:1 ratio of staple/scaffold given above

pH value adjustment

Buffer exchange

Use 2 tubes and one filter per sample

  • Insert the Amicon Ultra-0.5 device into one of the provided micro centrifuge tubes
  • Add 20 to 40 μL of sample to the Amicon Ultra filter device and fill it up to 500 μL with new buffer, cap the tube
  • Place capped filter device into the centrifuge rotor, aligning the cap strap toward the center of the rotor, counterbalance with a similar device.
  • Spin the device at 13k for 5 minutes
  • Separate the filter from tube and remove the buffer in the tube
  • Fill the filter again with 500 μL of the new buffer and put in the centrifuge rotor at 13k for 5 minutes
  • Repeat the procedure 1 - 3 times
  • Remove the assembled device from the centrifuge and separate the Amicon Ultra filter device from the micro centrifuge tube.
  • To recover the concentrated solute, place the Amicon Ultra Filter device upside down in the second clean micro centrifuge tube. Place in centrifuge, aligning open cap towards the center of the rotor; counterbalance with a similar device. Spin for 1 minutes at 3k to transfer the concentrated sample from the device to the tube. The ultra filtrate can be stored in the centrifuge tube.

Buffer preparation

  • TE buffer solution

for 1 l of 10x stock solution

- 100 ml 1M Tris-HCl pH 7.5 or 8.0

- 20 ml 0.5M EDTA pH 8.0 - 880 ml ddH2O


  • TBE buffer solution

for 1 liter of 5X TBE mix

- 54 g Tris Base

- 27.5 g Boric Acid

- 20 ml EDTA pH 8

- fill up with dH2O


  • PBS buffer solution

1x PBS buffer solution contains

- 137 mM NaCl

- 2.7 mM KCl

- 4.3 mM Na2HPO4

- 1.47 mM KH2PO4


  • Loading buffer

Loading buffer contains

- 6x Agarose Gel Loading buffer

- 30% Glycerol weight-to-volume in water

- 0.025% Xylene cyanol

- 0.025% Bromophenol blue


  • 1kb DNA Ladder

1kb DNA Ladder contains

- Gel Loading Dye Blue 6x from New Englan Biolab - 100 μl

- 1kB DNA-Ladder from New England Biolabs - 100 μl

- Distilled water - 400 μl

- Total volume - 600 μl

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