Biomod/2011/IITM/AcidArtists/Work diary

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Work Diary

August

Week 3

Made our first few structures using cadnano (i.e. hollow pipe and smiley)
Started our work on the Cadnano design of the agregator molecule

Week 4

Completed the final cadnano design of the large agregator molecule


September

Week 1

  • Completed the final cadnano design of the small agregator molecule.
  • Ordered the staples for our prototype model.
  • Calculated dilutions required. You can find the table <a href="https://docs.google.com/open?id=0BxVyX70RExMgMTkyNjRjNmMtNTVmYy00NzAxLTkyM2YtOWQzNWExNDI5ZGYy">here</a>

  • Week 2

    • Diluted the staples to a final concentration of 200uM and stored at 4°C.
    • Concentration measured using nanodrop.


    Week 3</h3>
    • Isolated the plasmid pGEX-4T1 from E-coli using phenol chloroform method and stored at 4°C
    • Purity was checked using Gel Electrophoresis
    • <img src="http://openwetware.org/images/3/3c/31_10_2011.jpg"/>
    • Concentration of Plasmid was measured using nanodrop

    • <h4>Week 4</h4>

      • 200uM concentration of staples stock was further diluted to 15uM.
      • Made Scaffold stock having 0.1uM concentration.
      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. -1 degree C 
      4.  Go to 2   repeat 65 times
      5. Hold at 4 degree C
      
      
      Gel Electrophoresis 
      2% agarose gel was made
      Result
      No bands corresponding to the structure seen
      

      October

      <h4>Week 1 </h4> 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 <h4>Week2 </h4> 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 <h4>Week 3 </h4>

      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 Running buffer made using Mg(OAc)2 instead of MgCl2 1% agarose gel was made Results No bands corresponding to the structure seen <h4>Week 4 </h4> 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) -1 degree C 4) Go to 2 repeat 30 times 5) T = 60 C for 05:00 mins 6) -0.5 degree celsius 7) Goto 5 repeat 71 times 8) Hold at 4 degree C

      GEL Electrophoresis Running buffer having Mg(OAc)2 was used 0.3% agarose gel was made Results Bands corresponding to the structure seen <img src="http://openwetware.org/images/b/ba/Final_gel_image.png"/>

      We have eluted the structure using montage® PCR Centrifugal filter devices and are waiting for TEM or an AFM image.


      Due to unavailability of an AFM or TEM image, we decided to test the structure using Absorbance readings at 260nm and the gel run along with a ladder.

      <img src="http://openwetware.org/images/d/dd/Reaction_mixture.png"/> All the samples will have overhangs of different lengths.

      Absorbance Readings: <img src="http://openwetware.org/images/a/a5/Table.png"/>

      From the above table, we notice that the absorbance increases after the annealing reaction. This is due to the relative increase in the number of single strand DNA. Before the formation of the structure, the Annealing reaction mixture had single stranded staples and double stranded scaffold whereas after the annealing reacting the mixture had less number of staples but more number of single stranded Scaffold. Thus we see an increase in the absorbance value, as single stranded DNA has more UV absorbance compared to Double stranded DNA.

      Although this is not a sufficient condition, it is one of the necessary conditions to prove the formation of the structure [the small agregator molecule]

      This is the absorbance of the initial sample:

      <img src="http://openwetware.org/images/8/81/123.png"/>

      This is the absorbance of the control:

      <img src="http://openwetware.org/images/f/fb/C22.JPG"/>


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