Biomod/2011/LMU/FolD'N'Assemble/Protocols
May
05-13-11(Timon, Alex, Ralf)
We wanted to find the best MgCl concentration and the best annealing time for our construct.
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(2b) |
8 | 55h | 10 | |
9 | 55h | 14 | |
10 | 55h | 18 |
Then we used electrophoresis in 2% agarose gel to test the samples
05-16-11 (Timon, Alex, Ralf)
Another test for best MgCl concentration and annealing time. We used a 2% agarose gel.
SampleNr. | Annealing time | MgCl(mM) | |
---|---|---|---|
1 | 55h | 16 | |
2 | 55h | 18 | |
3 | 55h | 16 | dimers(2b) |
4 | 55h | 18 | dimers(2b) |
5 | 55h | 16 | dimers(4b) |
6 | 55h | 18 | dimers(4b) |
05-17-11(Timon, Alex, Ralf)
We took first TEM (transmission electron microscope) pictures of our construct.
05-18-11(Timon, Alex, Ralf)
New samples for next week: 200μL, 18mM MgCl, 55h
05-23-11(Timon, Alex, Ralf)
We prepared buffers with different pH.
- pH 4,5
- pH 5
- pH 6
- pH 8
05-24-11(Alex, Ralf, Timon)
- prepared 3 samples of origamis for measuring its stability in a changing pH environment
- exchanged standard buffer with pH adjusted buffer using centrifugal filters
- 3 samples, ~24 h exposure to pH values of 4.5, 5 and 6, 1x TE buffer, 18mM MgCl2
05-25-11(Alex)
- made a gel with pH adjusted buffers, UV screenshot 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 (2% Agarose)
- 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 200nM
- started annealing new samples with i-motif
06-03-11(Timon)
Ran a gel(2% agaroses) with 3 samples
- monomers(cs)
- dimers(cs+4es)
- dimers + i-motif (cs+i-m)
06-07-11(Timon)
I Prepared new smples
- 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 (2% agarose)
- 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)
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. So i cut out the I-V bande(10 connections)and tried to change the i-motif by pH. Didnt 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
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.
08-19-11(Timon)
- New scaffold was produced in the lab. I ran a gel to compare the old and the new scaffold.
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