I made this…




                  Presents:


The Biological Preparation of
  Shotgun DNA Mapping
               By Antho...
Shotgun DNA Mapping in a Nutshell
                                                                           Library of
Pr...
Where do you start?
• Need genomic DNA
  from yeast
• Grow some yeast
• Extract the DNA
• Now we’re Koching



           ...
Yeast Cell
• Spheroplasting
• RNaseA-ing
• Phenol/Chloroform
  Extraction and Ethanol
  Precipitation



                 ...
Next Step
                                                              • Need digested plasmid
                          ...
The first of many gels
                                • Lanes:
                                  1: pRS413 uncut
        ...
Digested gDNA
• Lanes:
  1: Uncut gDNA
  2: gDNA cut with XhoI
  3: gDNA cut with XhoI (for
  redundancy)


              ...
Inserting DNA
• CIP – Calf Intestinal
  Phosphatase
• T4 DNA Ligase - ??? DNA
  Ligase




                             Te...
Making Clones
                             • Mix Competent E. coli
                               cells with plasmid DNA
 ...
1st and 2nd Transformation Tries




              A whole blown wad
Transformation Success?

                             E. Coli DNA


                             Extracted plasmid DNA



...
Double Digest and pBluescript




I was drunk when I took this picture
                                       I was drunk ...
Redoing with pBS
                           • Now that is definitely
                             some random genomic
    ...
Sequencing
• Involves some steps I
  don’t know
• Need to sequence so
  that when we unzip we
  can know what the
  correc...
Development of Tether Construct
         Part 1: PCR
                                  • Need:
                           ...
Tether Construction Part 2
                                                                        BstXI
                 ...
When it’s all done
     • More on next slide                                                                     gDNA
    ...
What I have now
What it looks like                What it should look like




                                both
      ...
Combine with Fragments
• Ligate the plasmid
  random fragments to
  the tethering construct
• Use flow chamber
  fluidics ...
None of you better look like this guy
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The Biological Preparation Of Shotgun DNA Mapping 5/15/09

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This is a talk I gave at the 1st KochLab Symposium. This talk provides a glimpse into my work at Osley Lab during the Spring Semester of 2009 at UNM. I present making random genomic fragments, cloning those fragments, and ligating the fragments to a construct that enables said fragments to be unzipped using an Optical Tweezer.

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The Biological Preparation Of Shotgun DNA Mapping 5/15/09

  1. 1. I made this… Presents: The Biological Preparation of Shotgun DNA Mapping By Anthony …and this
  2. 2. Shotgun DNA Mapping in a Nutshell Library of Procedure Simulated Curves Experimental Random Endonuclease Force Genomic fragment DNA Correct Match dsDNA anchor Step 1: Digest genome into Step 2: Unzip fragment and Step 3: Compare fragments record forces experimental forces to a library of simulated curves What this talk is about Austin is in there too
  3. 3. Where do you start? • Need genomic DNA from yeast • Grow some yeast • Extract the DNA • Now we’re Koching A blurry image of yeast cells
  4. 4. Yeast Cell • Spheroplasting • RNaseA-ing • Phenol/Chloroform Extraction and Ethanol Precipitation It’s foreign so it’s gotta be evil
  5. 5. Next Step • Need digested plasmid DNA and digested genomic DNA • Want to clone fragments – For sequencing – So we can unzip a lot of fragments Michael Bay’s next film… too late I already sold the rights
  6. 6. The first of many gels • Lanes: 1: pRS413 uncut 2: pRS cut with XhoI 3: pRS cut with NotI 4: pRS cut with BstXI 10kb length 5: genomic uncut DNA (gDNA) 6: gDNA cut with XhoI My archnemesis
  7. 7. Digested gDNA • Lanes: 1: Uncut gDNA 2: gDNA cut with XhoI 3: gDNA cut with XhoI (for redundancy) Get used to this, there is a lot more coming Making this was really annoying
  8. 8. Inserting DNA • CIP – Calf Intestinal Phosphatase • T4 DNA Ligase - ??? DNA Ligase Terminators can’t self terminate.
  9. 9. Making Clones • Mix Competent E. coli cells with plasmid DNA • E. coli readily replicates plasmid • Grow cells on petri dish • Cells grow into individual colonies One of them likes pizza • If plasmid has inserts then each colony is a separate insert
  10. 10. 1st and 2nd Transformation Tries A whole blown wad
  11. 11. Transformation Success? E. Coli DNA Extracted plasmid DNA This is all Koch’s fault
  12. 12. Double Digest and pBluescript I was drunk when I took this picture I was drunk when I slept with this one
  13. 13. Redoing with pBS • Now that is definitely some random genomic fragments • Top Image quick I like pink tape extraction • Bottom Image is good extraction
  14. 14. Sequencing • Involves some steps I don’t know • Need to sequence so that when we unzip we can know what the correct match is • Larry look away I thought it would be funny if I used a print screen of this slide for this slide.
  15. 15. Development of Tether Construct Part 1: PCR • Need: Template DNA Forward Primer Reverse Primer • We use pRL574, F834, and R1985 • The F834 primer has DIG (for glass attachment) • There is a BstXI site in Works just like rabbit mating amplified sequence.
  16. 16. Tether Construction Part 2 BstXI pRL fragment • Make an oligo that has BstXI site and is Biotinylated NotI • We made 2: NotI hairpin – One is a hairpin with a NotI site or – The other is two single stranded oligos with a SapI SapI site Top and bottom Annealed oligos • Remember our fragments have both NotI ends and NotI end SapI ends SapI end The sequel to Michael Bay’s movie Rights also already sold
  17. 17. When it’s all done • More on next slide gDNA plasmid Biotinylated fragment Digitylated fragment … Gel of Digested Fragments This is what skittles does to your DNA The quality of this image is a direct result of a computer from 1991
  18. 18. What I have now What it looks like What it should look like both fragment anchor 1991 strikes again 2009 artist rendition
  19. 19. Combine with Fragments • Ligate the plasmid random fragments to the tethering construct • Use flow chamber fluidics to prepare sample for tweezing • Wait 3 years for tweezer • Tweeze The bastard child of a koch and a wang Pronounced incorrectly
  20. 20. None of you better look like this guy

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