Biomod/2011/LMU/FolD'N'Assemble/Protocols
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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
(for the nanopill use 18mM MgCl2, m13/p8634 scaffold; the staple sequences can be found here[LINK])
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; optimal annealing times have again to be determined by experimentation and depend on the structure
(for the nanopill 55h hours proofed to be the most effective, acceptable results can already be achieved after 4h of annealing)
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 loading 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
- 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
pH value adjustment
Buffer exchange
Prepare the needed buffers with adjusted pH value
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 recipe
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
0.5xTBE Stock for 0.1 Liter (pH 8.2)
- Tris-base 5.4 g
- 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
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
Lab notebook
May
05-13-11(Timon, Alex, Ralf)
- 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.
| 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.
| 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)
- 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)
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)
I ran a gel (2% agarose)
- cs
- cs+4es
- cs+i-motif I+II
- 3x cs+i-motif I-V
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)
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)
- 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)
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)
- 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)
2% agarose gel to see if the new scaffold from the stock works. It did not.
08-19-11(Timon)
- 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)
- 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)
- 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
09-02-11(Timon)
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
- Prepared new samples
