Bio 181 Restriction Mapping Tutorial
You have cloned an unknown cDNA 3 kb long into the EcoRI site of vector plasmid
pBluescript (also 3 kb) . You need to prepare a restriction map of the cDNA insert to set up
your future experiments.
This is what you know about the ligated plasmid before you start:
MCS = multicloning site
Unknown
cDNA insert
3 kb
Left half of MCS:
EcoRI
PstI
SmaI
BamHI
XbaI
NotI
SacI
ampR
pBluescript
(vector)
3 kb
ori
Right half of MCS:
EcoRI
EcoRV
HindIII
ClaI
SalI
XhoI
ApaI
KpnI
The insert is cloned into the Eco RI site of the multicloning site (MCS/polylinker). You have
this restriction map of pBluescript, so you know what enzyme sites are on the extreme right
and left of the insert. As with all polylinker restriction sites, these sites are unique in the
vector.
You first cut the plasmid with all of these polylinker enzymes to see which of them cut in the
insert, and where the sites are relative to the site in the vector. You set up restriction digestion
for each of the enzymes and allow them to cut for one hour. After the incubation period, you
load them on a gel. You run the digestions from the left side on one gel and the right side on
another gel.
1
Left Side Enzymes
Marker
EcoRI
PstI
SmaI BamHI
XbaI NotI
SacI
Uncut
23.1
9.4
6.6
4.2
2.3
2.0
1.35
1.08
.87
.60
.31
.28
2
Right Side Enzymes
Marker
EcoRI EcoRV HindIII ClaI
SalI
XhoI ApaI KpnI
Uncut
23.1
9.4
6.6
4.2
2.3
2.0
1.35
1.08
.87
.60
.31
.28
3
To interpret the results, first determine whether each enzyme has a cut site within the insert
(you already know each enzyme cuts within the vector’s MCS). Based on whether the
enzyme as zero, one, or more than one site in the unknown cDNA insert, list the enzymes’
names in the appropriate category below. Next, list the sizes of the bands that they produce.
No Sites
Sma I
Xba I
Not I
EcoRV
Cla I
Sal I
Apa I
Single cutters
Pst I:
2.1 kb
3.9 kb
Sac I:
3.1
2.9
Multiple Sites
BamHI: 4.2
1.2
0.6
HindIII: 4.3
1.7
All produce
a single 6.0 kb
band
Xho I:
1.4
KpnI:
3.6
1.4
1.0
4.6
Bands must add
up to 6.0 kb (size
of whole plasmid)
Make estimates
of smallest bands
first; gel size precision
is poor at the higher
levels. Add up to 6.0
See maps at end of this document.
HINTS:
When estimating the sizes of the bands, note that the sizes of fragments larger than the 2.3 kb
marker are hard to estimate and therefore inaccurate. Rely more on the sizes of the smaller
bands and make sure that the sum of all the bands equals the total plasmid size (6.0 Kb).
•
•
•
If you see only one band and it's 6.0 Kb (3.0 Kb of insert and 3.0 Kb of vector) then put
that enzyme under "No Sites."
If you see two bands, put that enzyme under "Single Cutters". List the fragment sizes.
If you see more than two bands, put that enzyme under "Multiple Sites". List the
fragment sizes.
4
Mapping Single Cutters:
The enzymes that fall in the first category, “No Sites” in the cDNA are already mapped - they
don't exist in your insert. The digestion cut the plasmid only once, in the polylinker, and it was
linearized.
The enzymes that fall in the second category have one site inside your insert; two bands
resulted from one cut in the polylinker and one inside the insert of the circular plasmid. The
size of the smaller band is the distance from that enzyme's site in the polylinker to its
site in the insert. The larger band is the part of the insert left on the vector after the digestion
plus the vector plasmid (3.0 Kb).
For example, if enzyme X cuts in the right MCS and gives a 2.0 Kb band and a
4.0 Kb band, then the site for X in the insert is 2.0 Kb from the right side. If
enzyme Y cuts on the left side and gives a 0.5 Kb band and a 5.5 Kb band, then
the insert site for Y is 0.5 Kb from the left side:
Figure 2
On the back of this page, draw a restriction map of the “single” cutters. Label each
enzyme’s cut site in the vector’s MCS, and the location you deduced for the cut site in the
unknown cDNA insert.
5
Mapping Multiple Cutters:
The appearance of three bands in a lane on the gels on pages 2 & 3 means that the enzyme
cuts in three places. One cut is in the polylinker, which is part of the vector plasmid, so its
position is known - either on the left side of the insert or the right.
To map the two additional cut sites inside the insert is difficult. The location of one site can be
identified from the gel data you have:
The vector plasmid is 3.0 Kb. The largest fragment is larger than 3.0 Kb.
Since there are no other sites for this enzyme in the vector, the largest
fragment must contain the vector plus some of the insert. Therefore, the
second site is the size of the large fragment minus 3.0 kilobases from
the opposite side of the polylinker. That is, if the multiple cutter is on the
left and you have a 4.3 Kb fragment, then the second site is 1.3 kb from
the right side.
At this point, there are two possible positions for the final cut site that could account for the
two other DNA fragments seen on the gel.
For each of the “multiple cutter” enzymes, draw two maps. Each map should
definitively locate one of the cut sites within the insert, and should show one of the two
possible locations for the second cut site within the insert.
At present you do not have enough data to correctly identify the second cut site within the
insert. In order to locate it, the plasmid must be double cut: with the enzyme being mapped,
and with an enzyme that has a single, known cut site in the insert.
To pin down the location of the second cut site, choose an appropriate second enzyme. Do a
double digest, and look for disappearance of a band (cleavage into two smaller ones)
compared with the single digest. {Details in class}
6
1600
XhoI
1300
HindIII
1800
BamHI
1500
600
KpnI
2100
Pst I
3.0 kb insert
2900
Sac I
0/6000
EcoRI
PstI
SmaI
BamHI
XbaI
NotI
SacI
Whole plasmid 6.0
kb
3000
EcoRI
EcoRV
HindIII
ClaI
SalI
XhoI
ApaI
KpnI
3.0 kb vector
4500
Unspecified: 2nd BamHI site
; 2nd KpnI site
.
First, determine the two possible locations. For BamHI, these are at 600 & 1200
For KpnI, these are at 1600 & 2000
7
To determine which second site is correct:
Perform a double digest, using a second enzyme that will give you information about the region in
question (in this case, I chose HindIII for both; other choices are possible). Run single digest of
Bam or Kpn side-by-side on a gel with double digest and look for disappearance of a band.
Only pay attention to the two smaller bands which are created by the mystery cut site; the
large band, while it may be cleaved, is irrelevant for this analysis.
BamHI
BamHI + HindIII
BamHI + HindIII
4.2 kb
1.2 kb
0.6 kb
KpnI
(if Bam site is at 600)
(if Bam site is at 1200)
KpnI + HindIII
KpnI + HindIII
3.6 kb
1.4 kb
1.0 kb
0.7 kb
0.3 kb
(if Kpn site is at 1600)
(if Kpn site is at 2000)
8