Biomod/2011/LMU/FolD'N'Assemble/Protocols

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THE PROJECT
METHODS
PROTOCOLS
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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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