Microsoft Word - blank.doc

Chapter 20

If you do not destain for long enough, the whole gel

remains blue and the fragments cannot be

differentiated.

22. Place the destained gel on a light box or on an
    overhead projector.


23. Obtain a blank acetate sheet or plastic wrap and
    place it over the gel. Trace the pattern of bands onto
    the wrap or sheet. Be sure to draw a line where the
    bottom of each well starts.

Evidence

(a) Carefully measure the distance in millimetres that each

band migrated from the well origin. Copy Table 2into

your notebook and use it to record the distances.

Analysis

(b) Using the HindIII digestion as a marker, plot the

distance travelled (x-axis) versus the fragment

base-pair size (y-axis) on semilogarithmic paper.

Please note that the 23 130-base-pair fragment and

the 27 491-base-pair fragment do not resolve, but

instead travel as one band. Therefore, take an average

of their size for graphing purposes.

(c) Using interpolation, determine the fragment size of

the bands produced by digestion with BamHI and

EcoRI. Enter your calculated base-pair fragment sizes

into your table.

INVESTIGATION 20.2continued (d) Compare the calculated base-pair fragments to the

actual base-pair fragments. Use the restriction enzyme

map of bacteriophage lambda (Figure 1) to

determine the size of the actual band fragments for

each enzyme. Calculate the percentage error.

Evaluation

(e) What was the purpose of each tube? of the control?

(f ) Why do the smaller bands migrate faster than the

larger bands?

(g) Some bands that are close in size migrate together.

What measures may be taken to resolve bands close

in size?

(h) What purpose does the 1running buffer serve?

(i) Why must the gel be made using 1TBE buffer?

(j) During electrophoresis, bubbles are produced at the

anode and at the cathode. Explain why bubbles appear.

(k) Why must loading dye be added to the samples

before they are loaded into the wells of the gel?

(l) Notice on your gel that the larger fragments are

stained darker than the smaller fragments. Explain

why this is the case.

(m) Suggest possible sources of error in this procedure.

Indicate the effects of these sources of error on the

results.

Table 2 Distance Travelled by Each Band From the Well Origin

HindIII EcoRI BamHI

Actual Distance Actual Distance Calculated Actual Distance Calculated

fragment travelled fragment travelled fragment fragment travelled fragment

size (mm) size (mm) size size (mm) size

27 491

23 130

9 416

6 557

4 361

2 322

2 027

NEL Molecular Genetics 699