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

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===07-15-11(Timon)===
===07-15-11(Timon)===
[[Image:20110715 mono dim 4-10verbindungen ph8 scalt1.JPG|thumb|upright=1.5|06-20-11,left to right, Ladder 1kb, Scaffold,monomers, dimers, 4,6,8,10 i-motif connections TE pH8, old scaffold]]
[[Image:20110715 mono dim 4-10verbindungen ph8 scalt1.JPG|thumb|upright=1.5|07-15-11,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 works. It did not.
2% agarose gel to see if the new scaffold from the stock works. It did not.




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===08-19-11(Timon)===
===08-19-11(Timon)===
[[Image:20110819 sc8634 phagen.JPG|thumb|upright=1.5|08-19-11,left to right, Ladder 1kb,new scaffold1, new scaffold2, old scaffold, new phage, old phage]]


*New scaffold was produced in the lab. I ran a gel to compare the old and the new scaffold.  
*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.  




Revision as of 05:03, 10 October 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. 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, 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,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,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,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,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.




08-22-11(Alex)

08-22-11, 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, 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



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