Biomod/2012/TU Dresden/Nanosaurs/Lab book

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<a href="#recipes">Recipes</a> <a href="#protocols">Protocols</a> <a href="#downloads">Downloads</a>

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Recipes

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DNA origami recipes

FB 8 (1x / 10x)
Description Folding Buffer 8mM MgCl2: Buffer to be added before DNA origami assembly to create the necessary surrounding conditions for correct folding and assembly.
Recipe   1x 10x
  1M MgCl2 8µl 80µl
  1M Tris 5µl 50µl
  0,5M EDTA 2µl 20µl
  H2O 985µl 850µl
    1ml 1ml
Origami Assembly
Description Combines staple strands and scaffold in the necessary surrounding conditions. Staple strands have an excess of 7,5x in comparison to the scaffold to increase the chances of correct assembly.
Recipe 100nM p7560 single strand 10µl
  FB 8 10x 10µl
  Staple mix (≈500nM per oligo) 14,8µl
  H2O 65,2µl
    100µl


Staple mix
DescriptionThe certain mix of different staples (oligonucleotides) that are needed to
form different structures with their respective functions.
RecipeStaple typeOpenClosed guidedClosed not guided 
 corexxx 
 core_guideadjx x 
 guide_adj x  
 guide x  
 apt_locks xx 
 anchorxxx 
 catcherxxx 
 locks_nohangx  If only the part inside the DNA origami and no overhang of the locks is wanted.
 anchor_nohang   If anchor strands are not wanted.
 catcher_nohang   If catcher strands are not wanted.
 edge_stab_45   For labeling of the DNA origami.

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GUV recipes

SLB buffer
Description SLB (supported lipid bilayer) buffer is used as the solution for all the vesicle experiments.
Recipe 10mM HEPES
150mM NaCl
8mM Mg2++
DNA PAGE gel 12%
Recipe Polyacrylamid 3ml
H2O 5,894ml
TBE 10x 1ml
APS 100µl
TEMED 6µl
Ingredient
Stock Concentration
Amount (µl)
TdT Buffer   12
CoCl2   12
Oligonucleotide 100 µM 3
Alexa-dUTP conjugate/
Biotin-ddUTP conjugate		 1mM 3
Terminal Transferase   3
Water   27
Total   60
Procedure 1. Incubate at 370 C for 60 mins.
  2. Heat inactivate at 700 C for 15 mins.
  3. Cleanup with NucleoSpinExtract II kit.
  4. Measure the resultant concentration using <a href="http://www.nanodrop.com/?gclid=CPT5qOzCorMCFYq-zAodFA4Axw">NanoDrop</a>
<a id="protocols">

Protocols


Giant unilamellar vesicles (GUVs) formation

The method used to form the GUVs is “Vesicle Formation from emulsion”. This method consists in a controlled hydration of dried or nearly dried films of lipids deposited on a solid surface, in this case electrodes.

  1. Preparation of lipids solution

    2. The lipid mixture contains 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) in a 9:1 proportion in 1 mg/ml concentration. The solution is prepared with the organic solvent chloroform.

  2. Lipids spreading

    3. 6.5 µl of the lipid mixture is evenly spread in the surface of two little wires, the electrodes, which are previously cleaned by sonication cycles, washed with ethanol and dried.

  3. Solvent evaporation

    4. The lipid sample is left to dry over the electrodes inside an extractor for at least 15 min to evacuate the solvent of chloroform.

  4. Electroformation

    5. The electrodes are inserted in the lid of a plastic chamber that is filled with 3.5 ml of sucrose solution isomolar to the working buffer (HEPES, NaCl and w/o Mg2+). This solution hydrates the dry lipid sample to form the GUVs emulsion. The hydration is carried out in the presence of an electric field with a frequency of 10 Hz during 2 hours to electroformate the vesicles and of 2 Hz during 30 min to detached them from the electrodes.

<a rel="lightbox" href="http://www.openwetware.org/images/4/4b/GUVs_formation.png"><img src="http://www.openwetware.org/images/4/44/GUVs_formations_s.png"></a>
Representation of electroformation/ eletroswelling

Large unilamellar vesicles (LUVs) formation

The method employed consists of rehydratation and extrusion, whose detailed procedures are summarized as following:

  1. Preparation of lipids solution

    2. The lipid mixture of 1 mg/ml concentration contains 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) with 0%-10% DOPS volume. The solution is prepared with the organic solvent chloroform.

  2. Evaporation

    3. 25 µl of sample solution are distributed as evenly as possible in the interior surface of a crystal meanwhile it is blown-dry using nitrogen. To totally evacuate the solvent of chloroform, the vial is left during one hour inside a drier.

  3. Hydratation

    4. 500 µl of the working buffer (HEPES, NaCl and w/o Mg2+) is used to hydrate the dry lipid.

  4. Incubation

    5. The vials are left for 2 hours of incubation; mix the sample using a vortexer.

  5. Freeze/thaw

    6. The sample is immersed in liquid nitrogen followed by boiling water for 5 cycles totally.

  6. Extrusion

    7. Assemble the membrane inside the extruder as it follows:

    1. The Teflon piece is wetted with buffer.
    2. The 2 pieces of cellulose membrane are placed in a support in the center of the Teflon piece.
    3. Put the PC membrane with a pore size of 50-100 µm on the taller piece of the holder.
    4. A drop of buffer is placed on top of the membrane.
    5. The 2 pieces of Teflon holder are placed together.
    6. The syringe is filled with the lipid solution, and extrude at least 11 times.
    7. The extruded sample from the acceptor syringe is withdrawn. The solution should look clearer than it was before extrusion.
  7. Cleaning

    8. The Teflon piece, o-ring and syringe should be rinsed with copious 2-propanol and deionized water after use. Otherwise the residues will contaminate the next sample.

Aptamer Lock

Optimization Experiments

  1. Equi-molar quanities of unlabeled aptamer strands and Cy3 labeled aptamer locking strands were mixed to produce samples with 1 nM, 10 nM and 100 nM concentrations of Cy3 labeled strands.
  2. All the samples were made in SLB buffer (<a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#recipes">Lab Book -> Recipes</a>)
  3. The samples were then annealed in a PCR system and spectrophotometrically measured.
  4. Spectrophotometric measurements very done using 120 µl cuvettes in a spectrophotometer.
  5. The samples were excited at 510 nm and emission was measured at 564 nm.

Spectrophotometric Measurements

  1. All the spectrophotometric samples were prepared in SLB buffer (<a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#recipes">Lab Book -> Recipes</a>)
  2. A 10 nM concentration of the respective labeled/unlabeld lock/oligo was used
  3. When PDGF was used, it was always used in 10 times excess concentration than the oligos
  4. When either of the blockers were used, they were used in a 10 times excess concentration than the oligos
  5. All samples were incubated at room temperature for 24 Hrs.
  6. Spectrophotometric measurements very done using 120 µl cuvettes in a spectrophotometer.
  7. All the samples were excited at 510 nm and emission was measured at 624 nm

Gel Shift Assays

  1. All the gel samples were prepared in SLB buffer (<a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#recipes">Lab Book -> Recipes</a>)
  2. ~70 ng of DNA concentration was maintained for each sample.
  3. When PDGF was used, it was always used in 10 times excess concentration than the lock/oligos
  4. When either of the blockers were used, they were used in a 10 times excess concentration than the lock/oligos
  5. All samples were incubated at room temperature for 24 Hrs.
  6. Fermentas 6X Orange DNA Loading Dye was used with all the samples
  7. Fermentas O’RangeRuler 20 bp DNA Ladder was used
  8. Invitrogen, 4-20% TBE PAGE Gels were used and electrophoresis was performed for 40 minutes at 200 V

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