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

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HOME
THE PROJECT
METHODS
PROTOCOLS
LAB BOOK
RESULTS
TEAM

Timon Funck

Aleksej Belizki

Ralf Weidner

Miranda Roßmann

Verena Schüller

Prof. Tim Liedl



Lab notebook

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
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
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.




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
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
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
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
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, 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
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
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
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
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
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
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
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)
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)

  • 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)

09-09-11, Florescence image with i-motif dimers and different salt concentrations, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, momomers, imotif I-II, imotif I-V, dimers, imotif I-V 18 mM MgCl2, imotif I-V 10 mM MgCl2, imotif I-V 5 mM MgCl2, imotif I-V 50 mM MgCl2, imotif I-V 25 mM MgCl2, imotif I-V 50 mM NaCl, imotif I-V 50 mM MgCl2 in PBS, imotif I-II 18 mM MgCl2, imotif I-II 10 mM MgCl2, imotif I-II 5 mM MgCl2, imotif I-II 50 mM MgCl2, imotif I-II 25 mM MgCl2, imotif I-II 25 mM MgCl2, imotif I-II 50 mM NaCl, imotif I-II 50 mM NaCl in PBS
09-09-11, Florescence image with i-motif dimers and different salt concentrations, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, momomers, imotif I-II, imotif I-V, dimers, imotif I-V 18 mM MgCl2, imotif I-V 10 mM MgCl2, imotif I-V 5 mM MgCl2, imotif I-V 50 mM MgCl2, imotif I-V 25 mM MgCl2, imotif I-V 50 mM NaCl, imotif I-V 50 mM MgCl2 in PBS, imotif I-II 18 mM MgCl2, imotif I-II 10 mM MgCl2, imotif I-II 5 mM MgCl2, imotif I-II 50 mM MgCl2, imotif I-II 25 mM MgCl2, imotif I-II 25 mM MgCl2, imotif I-II 50 mM NaCl, imotif I-II 50 mM NaCl in PBS
  • 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)

09-12-11, Florescence image with i-motif dimers and different salt concentrations, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, imotif I-V 18 mM MgCl2 pH3, imotif I-V 10 mM MgCl2 pH 4.5, imotif I-V 5 mM MgCl2 pH 4.5, imotif I-V 50 mM MgCl2 pH 4.5, imotif I-V 25 mM MgCl2 pH 4.5, imotif I-V 50 mM NaCl pH 4.5, imotif I-V 50 mM MgCl2 in PBS pH 4.5
09-12-11, Florescence image with i-motif dimers and different salt concentrations, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, imotif I-V 18 mM MgCl2 pH3, imotif I-V 10 mM MgCl2 pH 4.5, imotif I-V 5 mM MgCl2 pH 4.5, imotif I-V 50 mM MgCl2 pH 4.5, imotif I-V 25 mM MgCl2 pH 4.5, imotif I-V 50 mM NaCl pH 4.5, imotif I-V 50 mM MgCl2 in PBS pH 4.5


  • repeated the gel electrophoresis from 09-09 with several samples, to get better images












09-16-11(Ralf, Miranda)

09-16-11, Florescence image with i-motif dimers, with different salt concentrations and buffers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, monomers, monomers PBS, monomers 5mM MgCl2, dimers, dimers 5 mM MgCl2, imotif I-II, imotif I-II PBS, imotif I-II MES, imotif I-III, imotif I-III PBS, imotif I-III MES, imotif I-IV, imotif I-IV PBS, imotif I-IV MES, imotif I-V, imotif I-V PBS, imotif I-V MES
09-16-11, Florescence image with i-motif dimers, with different salt concentrations and buffers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, monomers, monomers PBS, monomers 5mM MgCl2, dimers, dimers 5 mM MgCl2, imotif I-II, imotif I-II PBS, imotif I-II MES, imotif I-III, imotif I-III PBS, imotif I-III MES, imotif I-IV, imotif I-IV PBS, imotif I-IV MES, imotif I-V, imotif I-V PBS, imotif I-V MES
  • 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)

09-21-11, Florescence image with i-motif dimers, with different salt concentrations and buffers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, monomers, monomers PBS, monomers 5mM MgCl2, dimers, dimers 5 mM MgCl2, imotif I-II, imotif I-II PBS, imotif I-II MES, imotif I-III, imotif I-III PBS, imotif I-III MES, imotif I-IV, imotif I-IV PBS, imotif I-IV MES, imotif I-V, imotif I-V PBS, imotif I-V MES
09-21-11, Florescence image with i-motif dimers, with different salt concentrations and buffers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, monomers, monomers PBS, monomers 5mM MgCl2, dimers, dimers 5 mM MgCl2, imotif I-II, imotif I-II PBS, imotif I-II MES, imotif I-III, imotif I-III PBS, imotif I-III MES, imotif I-IV, imotif I-IV PBS, imotif I-IV MES, imotif I-V, imotif I-V PBS, imotif I-V MES


  • 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)

09-23-11, Florescence image with i-motif strands, 4% agarose gel,0.05x TBE running buffer at pH 4.5, 3h running time, left to right, Ladder 1kb, Scaffold, control strand, imotif II strand
09-23-11, Florescence image with i-motif strands, 4% agarose gel,0.05x TBE running buffer at pH 4.5, 3h running time, left to right, Ladder 1kb, Scaffold, control strand, imotif II strand
  • 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)

09-16-11, Laser scanner(left) and UV florescence(right)image of Cy5 strands attached to the monomer, 2% agarose gel, 3h running time, left to right, monomers, monomers after buffer exchange pH 8, monomers after buffer exchange pH 4.5
09-16-11, Laser scanner(left) and UV florescence(right)image of Cy5 strands attached to the monomer, 2% agarose gel, 3h running time, left to right, monomers, monomers after buffer exchange pH 8, monomers after buffer exchange pH 4.5
  • 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)

10-25-11, Florescence image with i-motif dimers, with different salt concentrations and buffers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, monomers, dimers, imotif I-II, imotif I-II PBS, imotif I-II PBS pH 4.5, imotif I-III, imotif I-III PBS, imotif I-III PBS pH 4.5, imotif I-V, imotif I-V PBS, imotif I-V PBS pH 4.5
10-25-11, Florescence image with i-motif dimers, with different salt concentrations and buffers, 2% agarose gel, 3h running time, left to right, Ladder 1kb, Scaffold, monomers, dimers, imotif I-II, imotif I-II PBS, imotif I-II PBS pH 4.5, imotif I-III, imotif I-III PBS, imotif I-III PBS pH 4.5, imotif I-V, imotif I-V PBS, imotif I-V PBS pH 4.5
  • ran a final gel to get best opening results of the dimers for results page














10-31-11(Ralf)

10-31-11, Laser scanner (right) and florescence(left) image with Cy5 loaded dimers, 2% agarose gel, 3h running time
10-31-11, Laser scanner (right) and florescence(left) image with Cy5 loaded dimers, 2% agarose gel, 3h running time


  • repeated experiment from 14-10 with fresh cy5 + ssDNA strands
  • good results, dimer is holding the cy5 dye
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