Gel Electrophoresis

The procedure for conducting electrophoresis and the principles it uses

An electric current is passed through agarose and smaller DNA fragments move faster and father from the origin than larger DNA fragments. Each band reperesents all of the DNA fragments of identical length. The dye used was Sybr Safe.

Function of buffer

Buffer is Tris-EDTA. chelates Mg+ to protect the DNA from the enzymatic degradation by DNAse.

In an electrical field, what direction (+or-) does DNA migrate? Why?

DNA moves toward the positive pole (anode) and away from the negative pole (cathode) because DNA is negative.

When doing gel electrophoresis, what would happen if you filled the gel with water instead of the gel buffer?

DNA would not move.

Function of sybr safe

Has a great affinity for double stranded DNA. Will glow under the fluorescent light.

Why did your transformed bacteria only fluoresce in the presence of arbinose, even though the results of your gel electrophoresis indicated that the GFP gene was present in all transformed colonies?

It only fluoresced in aribinose becasue arbinose sugar is used to activate the AraC operon which turns on the GFP gene, so no arabinose no glow. But, GFP was in all transformed bacteria because all the transformed bacteria got the plasmid which has the GFP gene. If got plasmid, GFP gene is present.

Function of agarose

Small fragments move more easily through agarose than large fragments, causing the smaller fragments to move faster and farther from the origin than the larger fragments.

How to estimate the size of unknown DNA fragments

So, you use a ruler to measure the distance the bands that you know the bp of to well. Then, you use a best fit line to calculate the amount of bps for the DNA fragment by having the distance from the well be y and the size of a DNA fragment be x. After you solve for x, you take the antilog of x and that will give you the base pairs.

Electrophoresis

The migration of charged substances in an electric field

DNA isolated and amplified from bacteria on LB/amp plates that did not glow tested positive for the presence of the pGLO gene. How can you explain that the gene was present however no GFP was being produced?

They still got the plasmid.

Function of UV light

Will show where the DNA strands are in the agarose gel. Will cause SYBR safe to light up like a freaking Christmas tree.

Sybr safe

a dye that fluoresces when exposed to ultraviolet light. Has a great affinity for double stranded DNA. Was incorporated into the agarose gel during its preparation and will attach to DNA fragments as they pass through the gel matrix

How did you produce the DNA products that were run in the gel?

amplified using polymerase chain reaction.

How to find how close your estimate is to the actual GFP gene size

found-actual/actual

Base pairs

fragment length, tris-EDTA

Buffer

maintains the sugar phosphate backbone and charge of DNA.

Agarose

the gel used in the experiment that an electric current is passed through so that DNA fragments will move according to size

Which PCR products from which of your plates tested positive for the presence of the GFP gene?

two and three.