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Supplemental information - Polymerase Chain Reaction (PCR) and microsatellites

For the prelab:
YOU MAY NEED TO DOWNLOAD ADOBE SHOCKWAVE PLAYER to use the PCR simulation below (or use the computers in lab to do the prelab)
PCR simulation (from the Dolan DNA Learning Center)
Main entry links for problem set:
NCBI home page
Virtual PCR website (Primer-Specificity-Checking)
To use this website, drop down "D.melanogas_nt" from the D.melanogaster list box.  Replace the default primers with your primers.  Click Submit.

If your primers are correct, the resulting page should list "big brain" as one of the target genes.

 
Other bib gene links:
NCBI Reference sequence for the Drosophila melanogaster bib gene
NCBI Sequence view for the bib gene
Drosophila melanogaster genome view

Sequence viewer 2.0 beta (replaces the version shown in the lab manual on p. 155)

Although it is annoying to have this change without warning, the new sequence viewer is actually less complicated and easier to understand.

Screenshot of new sequence viewer, zoomed out one level from default. 

To view the nucleotide sequence near the end of the gene where the microsatellite locus is located, click and drag the gene to the left so that the end of the red gene diagram is near the left side of the screen.  Click the enlarge button a number of times (always keeping the right end of the gene toward the left side of the screen) until you can enlarge it no further.  Click on the red bar.  You should see the detailed diagram shown below:

In this view not only are the single-letter abbreviations for the amino acids shown, but the particular nucleotides that comprise the codons that code for that amino acid are shown with the amino acid abbreviation.

 
Miscellaneous general interest links:
FlyBase report for the D. melanogaster Bar (B) gene
FlyBase report for the D. melanogaster black (b) gene

Thermus aquaticus and hot springs at Yellowstone Park
The Greatest Journey Ever Told: The Trail of Our DNA (National Geographic)

Color diagram for mini-review of Meiosis and Genetics

Electrophoresis

Gel simulator (courtesy of http://webphysics.davidson.edu/applets/biogel/biogel.html)

You can experiment with varying the gel density and electric field (i.e. through changing the voltage) using the simulator below.  This simulator mimics an agarose gel separating eight DNA fragments.  The fragment sizes (in kilobasepairs) are entered in the boxes at the top.  The agarose concentration and electric field are adjusted using the sliding controls at the bottom.  When you press compute, the simulator shows how far the bands would move from left to right during a fixed run time.  (The run time can not be varied.)  You will be asked to play with this simulator for the problem set.

 

Constructing a standard curve

On a particular gel, we do not know the relationship between protein weight and the distance traveled.  Thus a standard curve must be constructed, as discussed in the lab manual.  Proteins of known molecular weight are electrophoresed alongside with polypeptides of unknown molecular weight. Under most conditions, the electrophoretic mobility of a proteins is inversely proportional to the log of their molecular weight. The standard curve for electrophoresis is constructed by plotting the log of molecular weight of known proteins (y-axis) versus the distance migrated (x-axis) measured from the sample well.