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

HOME

THE PROJECT

METHODS

PROTOCOLS

LAB BOOK

RESULTS

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

Lab notebook

May

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

05-13-11, Florescence image with different annealing times and MgCl2 concentrations, 2% agarose gel, 3h running time, 70V, 0.5X TBE running buffer; From left to right: 1kb ladder, scaffold, sample 1-10

• 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.

05-16-11, Florescence image with varying MgCl2 concentrations and Dimers, 2% agarose gel, 3h running time, 70V, 0.5X TBE running buffer; From left to right: ladder, scaffold, sample 1-6

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)

05-30-11, Florescence image with samples at different pH values, 2% agarose gel, 3h running time, 70V, 0.5X TBE running buffer;left to right, Ladder 1kb, Scaffold, pH 8(control sample), pH 8, pH 6, pH 5, pH 4.5

• 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)

06-03-11, Florescence image with i-motif dimers, 2% agarose gel, 3h running timeleft to right, Ladder 1kb, Scaffold, momomers, dimers(4b), dimers with i-motif

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)

06-10-11,Florescence image with i-motif dimers, 2% agarose gel, 3h running timeleft to right, Ladder 1kb, Scaffold, momomers, dimers(4b), dimers with i-motif I+II, 3x dimers with i-motif I-V

I ran a gel (2% agarose)

• cs
• cs+4es
• cs+i-motif I+II
• 3x cs+i-motif I-V

06-10-11, Florescence image with i-motif dimers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, i-motif I-V pH 8, i-motif I-V pH 8 with buffer change, i-motif I-V pH 5 with buffer change

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)

06-17-11, Florescence image with i-motif dimers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, i-motif I-II, i-motif I-III, i-motif I-IV, i-motif I-V

06-17-11, Florescence image with i-motif dimers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, Monomers, i-motif I-V pH 5,6

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)

06-17-11, Florescence image with ES dimers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, (1):Monomers, 1x TE, untreated, pH 8, (2):Monomers, 1x TE, buffer exchange, pH 8 (3):Monomers, 1x TE, buffer exchange, pH 5, (4):Monomers, PB, buffer exchange, pH 5.3 (5):Monomers, PBS, buffer exchange, pH 5.8, (6):Dimers 2ES, 1x TE, buffer exchange, pH 8 (7):Dimers 2ES, 1x TE, buffer exchange, pH 5,(8):Monomers, PB, buffer exchange, pH 5.3 (9):Dimers 2ES, PBS, buffer exchange, pH 5.8

• 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)

06-20-11, Florescence image with i-motif dimers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, 4,6,8,10 i-motif connections TE pH8/ 4,6,8,10 i-motif connections Phosphat buffer pH 5,3/4,6,8,10 i-motif connections PBS pH 5,6

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)

07-11-11, Florescence image with i-motif dimers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, 4,6,8,10 i-motif connections TE pH8/ 4,6,8,10 i-motif connections Phosphat buffer pH 5,3/4,6,8,10 i-motif connections PBS pH 5,6

• 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)

07-15-11, Florescence image with i-motif dimers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold,monomers, dimers, 4,6,8,10 i-motif connections TE pH8, old scaffold

2% agarose gel to see if the new scaffold from the stock works. It did not.

08-19-11(Timon)

08-19-11, Florescence image with different scaffold solutions, 2% agarose gel, 3h running time, left to right, Ladder 1kb,new scaffold1, new scaffold2, old scaffold, new phage, old phage

• 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)

08-22-11, Florescence image with i-motif dimers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, momomers, dimers, dimers with i-motif I-II, I-III, I-IV and I-V

• 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)

08-25-11, Florescence image with i-motif dimers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, momomers, dimers, dimers with i-motif I-II (unmodified/pH4.5), I-III (unmodified/pH4.5), I-IV (unmodified/pH4.5) and I-V (unmodified/pH4.5)

• 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