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

05-13-11(Timon, Alex, Ralf)
We wanted to find the best MgCl concentration and the best annealing time for our construct.

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.

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 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.
 * 4,6,8,10 i-motif connections

New samples
 * 75μL i-motif I-II
 * 75μL i-motif I-III
 * 75μL i-motif I-IV
 * 75μL i-motif I-V



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)