Latest revision as of 16:58, 2 November 2011

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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)

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.

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)

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)

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)

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)

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)

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(Timon)

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)

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

08-19-11(Timon)

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)

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

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

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

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

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

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

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

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

repeated experiment from 14-10 with fresh cy5 + ssDNA strands

good results, dimer is holding the cy5 dye

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@@ Line 23: / Line 32: @@
 [[User:Ralf Weidner|<span style="color:black">Ralf Weidner</span>]]
+[[User:Miranda Roßmann|<span style="color:black">Miranda Roßmann</span>]]
 [http://www.softmatter.physik.uni-muenchen.de/tiki-index.php?page=CVschittler <span style="color:black">Verena Schüller</span>]
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-===May===
 ====05-13-11(Timon, Alex, Ralf)====
 [[Image:20110513 4h-24h-55h 10-14-18Mg.png| thumb |upright=1.5|05-13-11, Florescence image with different annealing times and MgCl2 concentrations, 2%
@@ Line 116: / Line 124: @@
 We took TEM (transmission electron microscope) images of the extracted samples.
-[Here be TEM images]
@@ Line 506: / Line 514: @@
 *Prepared new samples for gel electrophoresis
 ====09-07-11(Timon, Miranda)====
@@ Line 512: / Line 523: @@
 [gel image]
 ====09-09-11(Ralf)====
+[[Image:20110909.gif|thumb|upright=1.5|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
-[gel image]
+*Prepared new samples for gel electrophoresis
-*Prepared new samples for gel electrophoresis
 ====09-12-11(Timon)====
+[[Image:20110912.gif|thumb|upright=1.5|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
-[gel image]
 ====09-16-11(Ralf, Miranda)====
+[[Image:20110916.gif|thumb|upright=1.5|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
-[gel image]
+*extracted lane 10,13 and 19 for further TEM analysis
-*extracted lane 10,13 and 19 for further TEM analysis
 ====09-19-11(Timon)====
@@ Line 539: / Line 617: @@
 *took TEM images
-[TEM images]
 ====09-21-11(Timon)====
+[[Image:20110921.gif|thumb|upright=1.5|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
-[gel image]
+* extracted sample 14, 15, 16
-* extracted sample ?
 ====09-22-11(Timon)====
@@ Line 553: / Line 660: @@
 * took TEM images of samples from 21-09
-[TEM images]
 ====09-23-11(Alex)====
+[[Image:20110923.gif|thumb|upright=1.5|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
-* tried 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
-[gel image]
-* i-motif strand seems to have migrated with a different speed; could be an sign of i-motif formation
 ====09-26-11(Timon, Miranda)====
@@ Line 567: / Line 697: @@
 * tried dimerisation with new i-motif strands, these strands (IIa-Va) have more base mismatches than the old ones; could improve i-motif formation
-[gel image]
+* results: worse dimerisation, but no improvement of dimer opening
 ====10-14-11(Alex)====
@@ Line 573: / Line 705: @@
 * 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
-[gel image]
+* cy5 is barely visible on the laser scanner, need to increase concentration
-* cy5 is barely visible on the laser scanner, need to increase concentration
 ====10-14-11(Ralf)====
@@ Line 582: / Line 715: @@
 * no visible florescence; cy5 + dsDNA is probably bleached out, have to try with different dye
-[gel image]
 ====10-24-11(Alex)====
-*repeated experiment from 10-14 with higher concentrations of monomers
+[[Image:20110924.gif|thumb|upright=1.5|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
-[gel image]
-* images are better, this time
 ====10-25-11(Miranda)====
+[[Image:20111025.gif|thumb|upright=1.5|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
-[gel image]
 ====10-31-11(Ralf)====
+[[Image:20110931.gif|thumb|upright=1.5|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
-[image]
 * good results, dimer is holding the cy5 dye

Biomod/2011/LMU/FolD'N'Assemble/Labbook: Difference between revisions