Biomod/2011/LMU/FolD'N'Assemble/Labbook
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Lab notebook
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.
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)
- i-motif formation could be influenced by high salt concentration; the sequence could be shielded by the positive Mg2+ ions
We made new TE buffers with different salt concenctration:
- 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 for gel electrophoresis
09-07-11(Timon, Miranda)
- ran a gel, tried different salt concentrations, to see if the i-motif formation is related to it
[gel image]
09-09-11(Ralf)
- ran a gel, tried to further vary the salt concentration, this time also with NaCl
- Prepared new samples for gel electrophoresis
09-12-11(Timon)
- repeated the gel electrophoresis from 09-09 with several samples, to get better images
09-16-11(Ralf, Miranda)
- tried different buffer solutions(PBS/MES) to see if it would influence the i-motif formation
- extracted lane 10,13 and 19 for further TEM analysis
09-19-11(Timon)
- took TEM images
09-21-11(Timon)
- repeated gel from 09-16 to get better images
- extracted sample 14, 15, 16
09-22-11(Timon)
- took TEM images of samples from 21-09
09-23-11(Alex)
- tried a quick test to see if i-motif forms at all, ran a 4% gel at pH 4.5 and compared an i-motif strand with a regular strand of same length
- i-motif strand seems to have migrated with a different speed; could be an sign of i-motif formation
09-26-11(Timon, Miranda)
- tried dimerisation with new i-motif strands, these strands (IIa-Va) have more base mismatches than the old ones; could improve i-motif formation
- results: worse dimerisation, but no improvement of dimer opening
10-14-11(Alex)
- tried mechanism for loading the structure; strands with cy5 dye are attached to i-motif strands, when the pH value is lowered the load is automatically released; for proof-of-concept used the imotif strands on the monomers
- cy5 is barely visible on the laser scanner, need to increase concentration
10-14-11(Ralf)
- tried statistical loading of structure, by folding in a solution with high concentration of cy5 + dsDNA strand
- no visible florescence; cy5 + dsDNA is probably bleached out, have to try with different dye
10-24-11(Alex)
- repeated experiment from 10-14 with higher concentrations of monomers
- better images this time, thanks to higher concentrations; release of attached cy5 strands at pH 4.5 visible
10-25-11(Miranda)
- ran a final gel to get best opening results of the dimers for results page
10-31-11(Ralf)
- repeated experiment from 14-10 with fresh cy5 + ssDNA strands
- good results, dimer is holding the cy5 dye