Lab 7: Agarose Gel Electrophoresis Flashcards
What does spot plating do?
spot plating of serial dilutions allows for the direct count of CFUs through the number of colonies found growing in the spots.
What is gel electrophoresis?
the standard method used to separate, identify, and purify DNA fragments.
Describe the net negative charge.
the phosphate groups on the DNA backbone confer a net negative charge on the molecule. Thus, if a solution of DNA is placed into an electric field, the DNA molecules will migrate toward the positively charged electrode.
Describe gel electrophoresis.
the DNA fragments are forced to move through a porous matrix consisting of agarose. When DNA moves through the gel it must “snake” through the agarose, fitting through the pores.
What is the difference in movement between small fragments and large fragments?
Smaller fragments of DNA migrate more easily through the pores than larger fragments. Fragments will separate according to their sizes, with smaller fragments migrating faster and further from their origin than larger ones.
Describe the process of running the agarose gel.
Agarose is melted in a buffer and then allowed to cool in a casting tray to form a jello-like consistency. The casting tray contains a plastic comb that creates well in the agarose after it is removed. The hardened gel is then placed in a gel box with electrodes and covered in a buffer. A blue gel loading dye is added to the DNA samples before loading the samples into the wells of the gel. The DNA sample will sink to the bottom go the well instead of floating away. After the samples and ladder are loaded on the gel, a current is applied to the gel and the DNA begins to migrate into the gel and separates based on the sizes of the fragments. In order to visualize the DNA fragments generated by the PCR, you will add ethidium bromide (EtBr) to your gel.
What does the loading dye consist of?
glycerol and two tracking dyes: xylene cyanol and bromophenol blue. We will track the bromophenol blue front which migrates at appropriately the same rate as a 150 bp DNA fragment in a 2.0% agarose gel.
Why is EtBr harmful.
EtBr distorts the DNA upon binding, it can cause mutations in the DNA and is therefore a known (but weak) carcinogen.
What are the two kinds of percentage solutions?
Percentage by weight (w/v) for dissolving solids, and percentage by volume (v/v) for diluting concentrated % solutions.
Percentage by weight equation
(w/v) = grams of solute/100 ml of solution
If there are no bands on the gel, including the ladder, then
Ethidium Bromide was not added to the gel
If there are no sample bands and only ladder,
Component of Taq mix may have been degraded
If there are no bands present for a sample type then
One or both of the appropriate primers was not added to the master mix
