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

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*3 samples, ~24 h exposure to pH values of 4.5, 5 and 6, 1x TE buffer, 18mM MgCl2
*3 samples, ~24 h exposure to pH values of 4.5, 5 and 6, 1x TE buffer, 18mM MgCl2


<br style="clear:both;" clear="all" />
====05-25-11(Alex)====
====05-25-11(Alex)====
*made a gel with pH adjusted buffers, UV screenshot inconclusive, going to repeat experiment on Mo 05-30-11
*made a gel with pH adjusted buffers, UV screenshot inconclusive, going to repeat experiment on Mo 05-30-11


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===05-27-11(Alex)===
===05-27-11(Alex)===
*prepared samples to repeat the buffer pH adjusted gel, 150 μL, standard recipe
*prepared samples to repeat the buffer pH adjusted gel, 150 μL, standard recipe


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===05-30-11(Alex)===
===05-30-11(Alex)===
[[Image:Gel_ph_053011.png|thumb|upright=1.5|05-30-11,left to right, Ladder 1kb, Scaffold, pH 8(control sample), pH 8, pH 6, pH 5, pH 4.5]]
[[Image:Gel_ph_053011.png|thumb|upright=1.5|05-30-11,left to right, Ladder 1kb, Scaffold, pH 8(control sample), pH 8, pH 6, pH 5, pH 4.5]]
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*origami seems stable under changing pH conditions, extracted sample with pH 5 for further TEM analysis
*origami seems stable under changing pH conditions, extracted sample with pH 5 for further TEM analysis


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===05-31-11(Timon)===
===05-31-11(Timon)===
*made new staple mix for cs 200nM
*made new staple mix for cs 200nM
*started annealing new samples with i-motif
*started annealing new samples with i-motif


<br style="clear:both;" clear="all" />
===06-03-11(Timon)===
===06-03-11(Timon)===
[[Image:20110603 1.i-motif.JPG|thumb|upright=1.5|06-03-11,left to right, Ladder 1kb, Scaffold, momomers, dimers(4b), dimers with i-motif]]
[[Image:20110603 1.i-motif.JPG|thumb|upright=1.5|06-03-11,left to right, Ladder 1kb, Scaffold, momomers, dimers(4b), dimers with i-motif]]
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*dimers + i-motif (cs+i-m)
*dimers + i-motif (cs+i-m)


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===06-07-11(Timon)===
===06-07-11(Timon)===
I Prepared new smples
I Prepared new smples
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*100μL cs+i-motif II-V
*100μL cs+i-motif II-V


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===06-10-11(Timon)===
===06-10-11(Timon)===
[[Image:20110610 cs, cs+4es,imI+II,3ximI-V.JPG|thumb|upright=1.5|06-10-11,left to right, Ladder 1kb, Scaffold, momomers, dimers(4b), dimers with i-motif I+II, 3x dimers with i-motif I-V]]
[[Image:20110610 cs, cs+4es,imI+II,3ximI-V.JPG|thumb|upright=1.5|06-10-11,left to right, Ladder 1kb, Scaffold, momomers, dimers(4b), dimers with i-motif I+II, 3x dimers with i-motif I-V]]
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* i-motif I-V pH 5 with buffer change
* i-motif I-V pH 5 with buffer change


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===06-14-11(Timon)===
===06-14-11(Timon)===
New samples
New samples
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*25μL i-motif I-V
*25μL i-motif I-V


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===06-17-11(Timon)===
===06-17-11(Timon)===


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*75μL i-motif I-V
*75μL i-motif I-V


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===06-20-11(Timon)===
===06-20-11(Timon)===
[[Image:20110620 imotifconnectionsph.JPG|thumb|upright=1.5|06-20-11,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]]
[[Image:20110620 imotifconnectionsph.JPG|thumb|upright=1.5|06-20-11,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]]


Revision as of 05:45, 21 June 2011


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


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)

05-30-11,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 (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)

06-03-11,left to right, Ladder 1kb, Scaffold, momomers, dimers(4b), dimers with i-motif

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)

06-10-11,left 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,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 (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)

06-17-11,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,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. So i cut out the I-V bande(10 connections)and tried to change the i-motif by pH. Didnt work out either.

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)

06-20-11,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
  • 4,6,8,10 i-motif connections PBS pH 5,6
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