| EcoR I |
Hind III |
EcoR I / Hind III |
|
Tube #1
|
Tube #2
|
Tube #3
|
|
21226 [1]
|
23130 [1]
|
21226 [1]
|
|
7421 [4]
|
9416 [4]
|
5148 [7]
|
|
5804 [5]
|
6557 [7]
|
4973 [11]
|
|
5643 [3]
|
4361 [8]
|
4268 [6]
|
|
4878 [2]
|
2322 [3]
|
2037 [3]
|
|
3530 [6]
|
2037 [2]
|
1894 [2]
|
|
|
(564) [5]
|
1584 [10]
|
|
|
(125) [6]
|
1375 [5]
|
|
|
|
947 [4]
(831) [12]
(564) [8]
(125) [9]
|
[ ] indicates cleavage site position order. (Data from Table III)
( ) indicates fragment sizes too small to be imaged on the gel.
Student data should be expressed in Kilobase pairs (Kbp); thus, a fragment size
of 21,226 bp is expressed as 21.2 Kbp. Subsequent student map plots (Question
1) should agree with published data presented on your maps.
Question One:
Using the data from your Table, construct restriction maps for each of the
following digests:
lambda DNA EcoR I digest
lambda DNA Hind III digest
lambda DNA EcoR I / Hind III double digest
Teacher's note:
Completed maps are presented at the bottom of this page.
Published data is used. See Figure 5 for an example.
The restriction map for the combination lambda DNA digest (EcoR I/Hind III) is
drawn in the following manner: beginning at the left, find the first
restriction site location that occurs in EITHER individual map (it is position
21,226 in the EcoR I digest). Find the NEXT MUTUAL SITE LOCATION (it is 23,120
in the Hind III digest). To calculate the theoretical fragment length between
restriction sites 1 and 2, subtract 21,226 from 23,120, or 1,894. Thus there is
a fragment 1.89Kbp from the first site position to the second site position in
the restriction map of the double digest of lambda DNA using EcoR I/Hind III.
Now, find the third site position (it is 25,157 on Hind III). The distance
between it and the second site fragment is 2.03 Kbp. The data below summarizes
these calculations and also presents ranked fragments by position following
electrophoresis:
|
EcoR I / Hind III Digest
|
Site
No.
|
Fragment
size
|
(calculation)
|
Fragment
Position on Gel
|
|
(1)
|
212261
|
|
(2)
|
1894
|
[23120 - 21226]
|
7
|
|
(3)
|
2037
|
[25157 - 23120]
|
6
|
|
(4)
|
947
|
[26104 - 25157]
|
[10]
|
|
(5)
|
1375
|
[27479 - 26104]
|
9
|
|
(6)
|
4268
|
[31747 - 27479]
|
4
|
|
(7)
|
5148
|
[36895 - 31747]
|
2
|
|
(8)
|
564
|
[37459 - 36895]
|
[12]
|
|
(9)
|
125
|
[37584 - 37459]
|
[13]
|
|
(10)
|
1584
|
[39168 - 37584]
|
8
|
|
(11)
|
4973
|
[44141 - 39168]
|
3
|
|
[12]
|
831
|
[44972 - 44141]
|
[11]
|
|
(13)
|
3530
|
[48502 - 44972]
|
5
|
Expected (theoretical) gel fragment lengths are ranked by position (from
highest nucleotide number to lowest) as they appear on each gel lane. Values
given in [ ], above, denote fragments too small to be imaged on this percent
agarose gel.
Question Two:
From your restriction maps, identify the DNA bands that contain the genes for
head proteins and those that encode for cell lysis.
Teacher's notes:
- EcoR I:
- The DNA fragment length of 21226 bp (fragment 1) contains the genes for head proteins; fragment 6 (3530 bp) contains genes for cell lysis. (Fragment number in decending order as imaged on gel).
- Hind III:
- The DNA fragment length of 23120 bp (fragment 1) contains genes for head proteins; fragment 4 (4361 bp) contains genes for cell lysis. (Fragment number in decending order as imaged on gel.)
- EcoR I/Hind III: (lane 3)
- The DNA fragment length of 21226 bp (fragment 1) contains genes for head proteins; fragment 5 (3530 bp) contains genes for cell lysis. (Fragment number in decending order as imaged on gel.)
AE Partners Collection Index
