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

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PROTOCOLS

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TEAM Timon Funck Aleksej Belizki Ralf Weidner Verena Schüller Prof. Tim Liedl

May

05-13-11(Timon, Alex, Ralf)

05-13-11, From left to right: ladder, scaffolf, sample 1-10

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.

05-16-11, From left to right: ladder, scaffold, sample 1-6

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