Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
on an Agarose Gel
HC 70AL
Spring 2009
April 2, 2009
By Kristin Gill
How Do We Determine the Size of a PCR
Product?
12,216 bp
2,072 bp
1,500 bp
4,072 bp
3,054 bp
QuickTime
600 bp and a
decompressor
2,036 bp
QuickTime and a
are needed to see this p
decompressor
are needed
1,636 bpto see this picture.
1,018 bp
506/517 bp
396 bp 100 bp
How Can We Estimate the Size of the
Unknown DNA Fragment?
Measure how far each band traveled using a metric ruler (cm)
Example:
1000 1.5
900 1.7
800 1.9
700 2.2
600 2.7
500 3.7
400 4.9
Creating a Standard Curve
Plot the data points using an Excel program
1600
1400
1200
1000 Logarithmic
Trend line
800
600
400
200
0
0 2 4 6 8 10
How Is the Size of the Unknown DNA
Fragment Determined?
Measure the distance traveled by the unknown
fragment
Interpolate the size based on the standard curve
1600
1400
Example: 2 cm
1200
Fragment size: 875 bp
1000
875
800
600
400
200
0
0 2 4 6 8 10
Why Do You Need to Know the Size of the
PCR Product?
Sequencing!
The amount of DNA used in the sequencing
reaction is determined according to the size of
the fragment (in bp) recommended by Applied
Biosystems protocol
Size of PCR Product Amount of DNA Used in
(bp) Sequencing Reactions
100-200 1-3 ng
200-500 3-10 ng
500-1000 5-20 ng
1000-2000 10-40 ng