determining the presence/size of DNA fragments separated via gel electrophoresis
you completed your PCR amplification but now need to determine whether or not you have PCR products. To do this, you must visualize your products via gel electrophoresis.
Your PCR product bands are very small compared to those in other DNA experiments. The product band sizes in this lab are 455 bp for the plant primers and 200 bp for the GMO primers. We will use a concentrated gel matrix (3%) to ensure the bands don't "run" through the gel.
You will also load a molecular weight ruler (DNA standard) so that you have a reference to determine your product bands' sizes.
The gel will then be stained with Fast Blast stain to make the bands visible.
compare unknown fragments to the fragment position of your known ladder/marker sample to estimate unknown fragment size.
your known (marker/ladder)
was lambda DNA digested with HindIII. to the left are the fragment sizes measured in base pairs (bp) of this ladder...
your unknown fragments are fragments of lambda DNA digested with
PstI and EcoR1.
Below is a pic of those fragments alongside your known fragment ladder and your uncut lambda DNA control.
Welcome to Dr. Kate Brilakis' Learning Portal
gel electrophoresis
Band Visualization:
455 bp = Control: DNA annealed with plant primers
200 bp = GMO positive: DNA annealed with GMO primers
identifying the source of a DNA sample using restriction endonucleases
GMO lab part II:
separating DNA fragments produced via PCR