Read this lab handout, watch the prelab lecture recording, and then take the Prelab quiz
titled “Prelab Quiz Lab 13 – Gel Electrophoresis” as many times as necessary until you score 100%.
Remember that you earn points for completing the prelab quiz.
INTRODUCTION
This week, you will separate the bands produced in your previous restriction
digests by agarose gel electrophoresis. From the sizes of the bands on your gel, you will
draw a map of your plasmid indicating the position of all of the BamHI and XbaI
recognition sites. To load your sample on the gel, you will add some loading buffer to
your sample. This buffer contains Tris-HCl and EDTA to protect your DNA by
maintaining the pH and sequestering Mg+2. It also contains glycerol (denser than water)
to help your sample sink to the bottom of the well, a tracking dye so that you can see
your sample in the well and watch it move through the gel (this dye is also negatively
charged so it moves through the gel as well, and a fluorescent dye that binds to the DNA
backbone. When your gel is exposed to UV light, this dye will fluoresce blue and
indicate that location of your DNA within the gel. Your instructor will load a DNA
standard in the first lane of your gel that can be used as a reference to determine the
sizes of the bands in your lanes. The sizes of the pieces of DNA in this standard are
shown in Figure 1 to the right. Notice that the 1 Kb band is darker than the others. Use
this darker band to determine the sizes of each band on your gel since some of the bands
on the bottom may run off the gel and some on the top may not separate from each other.
REQUIRED LAB NOTEBOOK ENTRY
o Title and date
o Purpose statement
o Data – Picture of your gel or a list of the sizes of DNA that you got
from each digest
Label your lanes (BamHI / XbaI / BamHI&Xba1)
Label the sizes of pieces of DNA in the ladder
o Analysis/Conclusion – Draw a map of the plasmid showing all of the
BamHI and XbaI recognition sites and the distances between them.
0.25 Kb
0.5 Kb
0.7 Kb
1.0 Kb
1.5 Kb
2.0 Kb
2.5 Kb
3.0 Kb
12.0 Kb
10.0 Kb
8.0 Kb
6.0 Kb
5.0 Kb
4.0 Kb
Figure 1
PROCEDURE
- Add _ µl of 6x loading buffer (6x LB) to your 20 μl of DNA digest from last week so that
the FINAL concentration is 1x.
6x LB = 60 mM Tris-HCl pH 8.0
6 mM EDTA
30% Glycerol
Tracking dye
Fluorescent DNA binding dye - Mix by flicking.
- Spin briefly to collect liquid at the bottom of the tube in a mini microfuge.
- Load 20 ul of your sample into 1 well of a 1% agarose gel.
- Run gel at 150 volts for about 1 hour.
- Visualize the fluorescent dye bound to your DNA by illuminating the gel with UV light to
determine the number of bands produced by each digest and the size of each piece produced. - Take a picture of your gel for you lab notebook and analysis.
ANALYSIS
Compare your bands to the DNA standard in lane 1 of your gel to determine the sizes of the
bands for each of your digests. The sizes of the standards are shown in Figure 1 (identify the standards
by first finding the 1 kb band as it is the darkest/thickest band and then identify the others from there).
From these sizes, draw a map of your plasmid showing all BamHI and XbaI sites and the distances
between them.
EVALUATION - Create a figure of your gel and label the bands in the ladder, your lanes (including which
enzymes were used in each lane) and the sizes of your bands. - Create a map of the plasmid you purified based on the data from your gel on question 5 of
the last page of this handout. - Answer the questions on the following page.
- Submit your figure and your completed last page of this handout to its assignment
submission folder in D2L Brightspace.
Genetics BIOL 262 Lab
Name _____ _________
Lab 13. Gel Electrophoresis & Plasmid Map Lab
Due at the end of lab
- How much of a 10x buffer should be added to 900ml of water to make a 1x solution?
- How much of a 5x buffer and how much water should be added to make 1 L of a 1x solution?
- How much of a 100x buffer and how much water should be combined to make 50 ml of a 1x
solution? - You want to make a solution that is 1x TE AND 10% glycerol in the same solution. You have
the following stock solutions:
10x TE
50% Glycerol
Water
How much of each should be combined to make 100 mls? - After viewing and analyzing your gel, draw a map of your plasmid showing all BamHI and XbaI
sites and the distances between them.