Biomod/2011/IITM/AcidArtists/Work diary: Difference between revisions

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<div id="contents_stuff">
<h1>Work Diary</h1>
<h3>August</h3>
<h4>Week 3</h4>
<p>Made our first few structures using cadnano  (i.e. hollow pipe and smiley)<br/>
Started our work on the Cadnano design of the aggregator molecule
</p>
<h4>Week 4</h4>
<p>Completed the final cadnano design of the large aggregator molecule</p><br/>
<h3>September</h3>
<h4>Week 1</h4>
<ul><li>Completed the final cadnano design of the small aggregator molecule.</li>
<li>Ordered the staples for our prototype model.</li>
<li>Calculated dilutions required. You can find the table <a href="https://docs.google.com/open?id=0BxVyX70RExMgMTkyNjRjNmMtNTVmYy00NzAxLTkyM2YtOWQzNWExNDI5ZGYy">here</a></li>
<br/>
<h4>Week 2</h4>
<ul><li>Dilluted the staples to a final concentration of 200uM and stored at 4°C.</li>
<li>Concentration measured using nanodrop.</li></ul>
<br/>
<h4>Week 3</h3>
<ul><li>Isolated the plasmid pGEX-4T1 from E-coli using phenol chloroform method and stored at 4°C</li>
<li>Purity was checked using Gel Electrophoresis<li>
<img src="http://openwetware.org/images/a/a6/Plasmid_gel_image.jpg"/>
<li>Concentration of Plasmid was measured using nanodrop</li>
<br/>
<h4>Week 4</h4>
<ul><li>200uM concentration of staples stock was further diluted to 15uM.</li>
<li>Made Scaffold stock having 0.1uM concentration.</li>
</ul>
<pre>First annealing reaction set in a PCR machine:
10x folding Buffer        = 5ul
Scaffold                          = 10ul
1ul of each staple *16  = 16ul                        (Assuming Staples Stock Concentration is 15ul)
DEPC water              = 19ul
Concentration of PCR reaction Mix
Staples  300nM each
Scaffold 20nM
PCR Program
1. T= 95 for 05:00min
2. T=90 for 01:00 min
3. -10C
4.  Go to 2  repeat 65 times
5. Hold at 4 0C
Gel Electrophoresis
2% agarose gel was made
Result
No bands corresponding to the structure seen
October
Week 1
Made the PCR reaction mixture having the same concentration as that of the previous one
Gel Electrophoresis
1% agarose gel was made
Result
No bands corresponding to the structure seen
Week2
Made the new scaffold stock having 0.25uM concentration
Made the PCR reaction mixture having the following concentration
10x folding Buffer        = 5ul
Scaffold                          = 8ul
2ul of each staple *16  = 32ul                        (Assuming Staples Stock Concentration is 15ul)
DEPC water              = 5ul
Concentration of PCR reaction Mix
Staples 600nM each
Scaffold 40nM
Gel Electrophoresis
1% agarose gel was made
Results:
No bands corresponding to the structure seen
Week 3
Made the PCR reaction mixture having the following concentration
10x folding Buffer        = 5ul
Scaffold                          = 8ul
2ul of each staple *16  = 32ul                        (Assuming Staples Stock Concentration is 15ul)
DEPC water              = 5ul
Concentration of PCR reaction Mix
Staples 600nM each
Scaffold 40nM
Gel Electrophoresis
New Runnig buffer made using Mg(OAc)2 instead of MgCl2
1% agarose gel was made
Results
No bands corresponding to the structure seen
Week 4
Made the PCR reaction mixture having the following concentration
10x folding Buffer        = 5ul
Scaffold                          = 8ul
2ul of each staple *16  = 32ul                        (Assuming Staples Stock Concentration is 15ul)
DEPC water              = 5ul
Concentration of PCR reaction Mix
Staples 600nM each
Scaffold 40nM
PCR Program used
1) T= 95 for 05:00min
2) T=90 for 04:00 min
3) -10C
4)  Go to 2    repeat 30  times
5)  T = 60 C for 05:00 mins
6) -0.5 degree celcius
7) Goto 5 repeat 71 times
8) Hold at 4 0C
GEL Electrophoresis
Runnig buffer having  Mg(OAc)2  was used
0.3% agarose gel was made
Results
Bands corresponding to the structure seen</pre>


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<div id="contents_stuff"> <h1>Work Diary</h1> <h3>August</h3> <h4>Week 3</h4> <p>Made our first few structures using cadnano (i.e. hollow pipe and smiley)<br/> Started our work on the Cadnano design of the aggregator molecule </p> <h4>Week 4</h4> <p>Completed the final cadnano design of the large aggregator molecule</p><br/> <h3>September</h3> <h4>Week 1</h4> <ul><li>Completed the final cadnano design of the small aggregator molecule.</li> <li>Ordered the staples for our prototype model.</li> <li>Calculated dilutions required. You can find the table <a href="https://docs.google.com/open?id=0BxVyX70RExMgMTkyNjRjNmMtNTVmYy00NzAxLTkyM2YtOWQzNWExNDI5ZGYy">here</a></li>

<br/> <h4>Week 2</h4> <ul><li>Dilluted the staples to a final concentration of 200uM and stored at 4°C.</li> <li>Concentration measured using nanodrop.</li></ul>

<br/> <h4>Week 3</h3> <ul><li>Isolated the plasmid pGEX-4T1 from E-coli using phenol chloroform method and stored at 4°C</li> <li>Purity was checked using Gel Electrophoresis<li> <img src="http://openwetware.org/images/a/a6/Plasmid_gel_image.jpg"/> <li>Concentration of Plasmid was measured using nanodrop</li>

<br/> <h4>Week 4</h4> <ul><li>200uM concentration of staples stock was further diluted to 15uM.</li> <li>Made Scaffold stock having 0.1uM concentration.</li> </ul>

<pre>First annealing reaction set in a PCR machine: 10x folding Buffer = 5ul Scaffold = 10ul 1ul of each staple *16 = 16ul (Assuming Staples Stock Concentration is 15ul) DEPC water = 19ul Concentration of PCR reaction Mix Staples 300nM each 

Scaffold 20nM

PCR Program 1. T= 95 for 05:00min 2. T=90 for 01:00 min 3. -10C 4. Go to 2 repeat 65 times 5. Hold at 4 0C


Gel Electrophoresis 2% agarose gel was made Result No bands corresponding to the structure seen October Week 1 Made the PCR reaction mixture having the same concentration as that of the previous one 

Gel Electrophoresis

1% agarose gel was made Result No bands corresponding to the structure seen Week2 Made the new scaffold stock having 0.25uM concentration Made the PCR reaction mixture having the following concentration

10x folding Buffer = 5ul Scaffold = 8ul 2ul of each staple *16 = 32ul (Assuming Staples Stock Concentration is 15ul) DEPC water = 5ul Concentration of PCR reaction Mix Staples 600nM each 

Scaffold 40nM

Gel Electrophoresis 1% agarose gel was made Results: No bands corresponding to the structure seen Week 3 

Made the PCR reaction mixture having the following concentration

10x folding Buffer = 5ul Scaffold = 8ul 2ul of each staple *16 = 32ul (Assuming Staples Stock Concentration is 15ul) DEPC water = 5ul Concentration of PCR reaction Mix Staples 600nM each 

Scaffold 40nM

Gel Electrophoresis New Runnig buffer made using Mg(OAc)2 instead of MgCl2 1% agarose gel was made Results No bands corresponding to the structure seen Week 4 Made the PCR reaction mixture having the following concentration 10x folding Buffer = 5ul Scaffold = 8ul 2ul of each staple *16 = 32ul (Assuming Staples Stock Concentration is 15ul) DEPC water = 5ul Concentration of PCR reaction Mix Staples 600nM each 

Scaffold 40nM

PCR Program used 1) T= 95 for 05:00min 2) T=90 for 04:00 min 3) -10C 4) Go to 2 repeat 30 times 5) T = 60 C for 05:00 mins 6) -0.5 degree celcius 7) Goto 5 repeat 71 times 8) Hold at 4 0C

GEL Electrophoresis Runnig buffer having Mg(OAc)2 was used 0.3% agarose gel was made Results Bands corresponding to the structure seen</pre>

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