Relative Activity of Restriction Enzymes in Promega 10X Buffers
The 10X Reaction Buffer supplied with each restriction enzyme is optimized to give 100% activity. In many cases, good activity is also obtained using one of the 4-CORE® 10X Buffers (Cat.# R9921). Table 3.1 may be used to select the best buffer for digestions with multiple restriction enzymes. Enzyme activity is expressed as a percent of the activity obtained with the optimized buffer for each enzyme.
| Promega Enzyme |
Buffer Supplied with Enzyme |
4 CORE® Buffers |
MULTI-CORE™ Buffer |
Enzyme
Assay
Temperature |
| A |
B |
C |
D |
| AatII |
J |
50-75% |
10-25% |
<10% |
<10% |
<10% |
37°C |
| AccI |
G |
50-75% |
25-50% |
25-50% |
10-25% |
25-50% |
37°C |
| AccIII |
F |
<10% |
10-25% |
25-50% |
25-50% |
<10% |
65°C |
| Acc65I |
D |
10-25% |
50-75% |
75-100% |
100% |
100% |
37°C |
| AccB7I |
E |
10-25% |
50-75% |
100%** |
<10% |
100% |
37°C |
| AgeI |
K |
25-50% |
25-50% |
25-50% |
50-75% |
100% |
37°C |
| AluI |
B |
75-100% |
100% |
75-100% |
10-25% |
10-25% |
37°C |
| Alw26I |
C |
10-25% |
25-50% |
100% |
10-25% |
75-100% |
37°C |
| Alw44I |
C |
<10% |
25-50% |
100% |
25-50% |
100% |
37°C |
| ApaI |
A |
100% |
50-75% |
50-75% |
<10% |
75-100% |
37°C |
| AvaI |
B |
10-25% |
100% |
50-75% |
25-50% |
<10% |
37°C |
| AvaII |
C |
50-75% |
50-75% |
100% |
25-50% |
25-50% |
37°C |
| Bal I |
G |
10-25% |
<10% |
<10% |
<10% |
<10% |
37°C |
| BamHI |
E |
75-100% ** |
75-100% |
75-100% |
50-75% |
75-100% |
37°C |
| BanI |
G |
25-50% |
25-50% |
10-25% |
<10% |
100% |
50°C |
| BanII |
E |
75-100% |
75-100% |
75-100% |
25-50% |
100% |
37°C |
| BbuI |
A |
100% |
75-100% |
75-100% |
<10% |
100% |
37°C |
| Bcl I |
C |
10-25% |
75-100% |
100% |
50-75% |
10-25% |
50°C |
| Bgl I |
D |
10-25% |
25-50% |
75-100% |
100% |
100% |
37°C |
| Bgl II |
D |
25-50% |
75-100% |
75-100% |
100% |
<10% |
37°C |
| BsaMI |
D |
10-25% |
25-50% |
50-75% |
100% |
25-50% |
65°C |
| BsaOI |
C |
10-25% |
50-75% |
100% |
25-50% |
100% |
50°C |
| Bsp I286I |
A |
100% |
50-75% |
25-50% |
10-25% |
75-100% |
37°C |
| BsrBRI |
H |
10-25% |
50-75%** |
100%** |
50-75% |
100% |
65°C |
| BsrSI |
D |
10-25% |
25-50% |
10-25% |
100% |
100% |
65°C |
| BssH II |
H |
75-100% |
50-75% |
75-100% |
50-75% |
75-100% |
50°C |
| Bst71 I |
D |
10-25% |
25-50% |
25-50% |
100% |
10-25% |
50°C |
| Bst98I |
D |
<10% |
10-25% |
10-25% |
100% |
25-50% |
37°C |
| BstEII |
D |
25-50% |
50-75% |
50-75% |
100% |
100% |
60°C |
| BstOI |
C |
10-25% |
25-50% |
100% |
25-50% |
<10% |
60°C |
| BstXI |
D |
<10% |
10-25% |
25-50% |
100% |
10-25% |
50°C |
| BstZI |
D |
<10% |
<10% |
10-25% |
100% |
10-25% |
50°C |
| Bsu36I |
E |
<10% |
25-50% |
50-75% |
25-50% |
50-75% |
37°C |
| CfoI |
B |
75-100% |
100% |
75-100% |
25-50% |
100% |
37°C |
| ClaI |
C |
75-100% |
75-100% |
100% |
75-100% |
100% |
37°C |
| CspI |
K |
<10% |
10-25% |
25-50% |
50-75% |
10-25% |
30°C |
| Csp45I |
B |
25-50% |
100% |
50-75% |
25-50% |
50-75% |
37°C |
| DdeI |
D |
25-50% |
25-50% |
50-75% |
100% |
25-50% |
37°C |
| DpnI |
B |
50-75% |
100% |
75-100% |
50-75% |
100% |
37°C |
| DraI |
B |
75-100% |
100% |
75-100% |
50-75% |
25-50% |
37°C |
| EclHKI |
E |
<10% |
<10% |
75-100% |
10-25% |
50-75% |
37°C |
| Eco47III |
D |
<10% |
25-50% |
50-75% |
100% |
25-50% |
37°C |
| Eco52I |
L |
<10% |
<10% |
10-25% |
25-50% |
<10% |
37°C |
| EcoICRI |
B |
10-25% |
100% |
75-100% |
<10% |
100% |
37°C |
| EcoRI |
H |
25-50% |
50-75% |
50-75% |
50-75% |
100% |
37°C |
| EcoRV |
D |
10-25% |
25-50% |
50-75% |
100% |
100% |
37°C |
| FokI |
B |
75-100% |
100% |
75-100% |
25-50% |
50-75% |
37°C |
| HaeII |
B |
50-75% |
100% |
50-75% |
10-25% |
100% |
37°C |
| HaeIII |
C |
75-100% |
75-100% |
100% |
50-75% |
100% |
37°C |
| HhaI |
C |
50-75% |
75-100% |
100% |
50-75% |
75-100% |
37°C |
| HincII |
B |
25-50% |
100% |
25-50% |
50-75% |
100% |
37°C |
| HindIII |
E |
25-50% |
100% |
75-100% |
10-25% |
50-75% |
37°C |
| HinfI |
B |
50-75% |
100% |
75-100% |
75-100% |
50-75% |
37°C |
| HpaI |
J |
25-50% |
50-75% |
25-50% |
10-25% |
100% |
37°C |
| HpaII |
A |
100% |
50-75% |
50-75% |
10-25% |
100% |
37°C |
| Hsp92I |
F |
10-25% |
75-100% |
50-75% |
25-50% |
10-25% |
37°C |
| Hsp92II |
K |
10-25% |
25-50% |
25-50% |
<10% |
<10% |
37°C |
| I-PpoI |
I-Ppo I |
10-25% |
25-50% |
25-50% |
25-50% |
- |
37°C |
| KpnI |
J |
100%** |
25-50% |
25-50% |
<10% |
75-100% |
37°C |
| MboI |
C |
10-25% |
75-100% |
100% |
50-75% |
<10% |
37°C |
| MboII |
B |
10-25% |
100% |
50-75% |
75-100% |
100% |
37°C |
| MluI |
D |
10-25% |
25-50% |
50-75% |
100% |
10-25% |
37°C |
| MspI |
B |
75-100% |
100% |
75-100% |
25-50% |
25-50% |
37°C |
| MspA1I |
C |
25-50% |
100%** |
100% |
10-25% |
100% |
37°C |
| NaeI |
A |
100% |
50-75% |
25-50% |
<10% |
50-75% |
37°C |
| NarI |
G |
75-100% |
50-75% |
75-100% |
25-50% |
50-75% |
37°C |
| NciI |
B |
100%** |
100% |
25-50% |
25-50% |
50-75% |
37°C |
| NcoI |
D |
50-75% |
75-100% |
75-100% |
100% |
75-100% |
37°C |
| NdeI |
D |
<10% |
<10% |
25-50% |
100% |
25-50% |
37°C |
| Nde II |
D |
<10% |
<10% |
10-25% |
100% |
25-50% |
37°C |
| NgoMIV |
MULTI-
CORE™ |
100%** |
100%** |
100%** |
<10% |
100% |
37°C |
| NheI |
B |
75-100% |
100% |
75-100% |
10-25% |
100% |
37°C |
| Not I |
D |
<10% |
10-25% |
25-50% |
100% |
25-50% |
37°C |
| NruI |
K |
<10% |
<10% |
<10% |
50-75% |
10-25% |
37°C |
| NsiI |
D |
10-25% |
50-75% |
50-75% |
100% |
10-25% |
37°C |
| PstI |
H |
10-25% |
50-75% |
50-75% |
50-75% |
25-50% |
37°C |
| PvuI |
D |
10-25% |
25-50% |
50-75% |
100% |
<10% |
37°C |
| PvuII |
B |
25-50% |
100% |
50-75% |
25-50% |
50-75% |
37°C |
| RsaI |
C |
75-100% |
75-100% |
100% |
<10% |
<10% |
37°C |
| SacI |
J |
75-100% |
25-50% |
25-50% |
<10% |
100% |
37°C |
| SacII |
C |
100%* |
50-75% |
100% |
50-75% |
<10% |
37°C |
| Sal I |
D |
<10% |
10-25% |
25-50% |
100% |
<10% |
37°C |
| Sau3AI |
B |
25-50% |
100% |
75-100% |
<10% |
100% |
37°C |
| Sau96I |
C |
25-50% |
25-50% |
100% |
50-75% |
50-75% |
37°C |
| ScaI |
K |
<10% |
100%** |
50-75% |
75-100% |
10-25% |
37°C |
| SfiI |
B |
75-100% |
100% |
75-100% |
25-50% |
75-100% |
50°C |
| SgfI (a) |
C |
25-50% |
25-50% |
100% |
<10% |
<10% |
37°C |
| SinI |
A |
100% |
75-100% |
50-75% |
10-25% |
100% |
37°C |
| SmaI |
J |
<10% |
<10% |
10% |
<10% |
100% |
25°C |
| SnaBI |
B |
50-75% |
100% |
50-75% |
<10% |
100% |
37°C |
| SpeI |
B |
75-100% |
100% |
75-100% |
75-100% |
100% |
37°C |
| SphI |
K |
75-100% |
75-100%** |
100% |
75-100% |
10-25% |
37°C |
| SspI |
E |
10-25% |
50-75% |
50-75% |
75-100% |
50-75% |
37°C |
| StuI |
B |
75-100% |
100% |
75-100% |
50-75% |
50-75% |
37°C |
| StyI |
F |
25-50% |
75-100% |
75-100% |
75-100% |
<10% |
37°C |
| TaqI |
E |
10-25% |
25-50% |
50-75% |
50-75% |
100% |
65°C |
| Tru9I |
F |
75-100% |
50-75% |
75-100% |
25-50% |
25-50% |
65°C |
| Tth111 I |
B |
50-75% |
100% |
75-100% |
25-50% |
100% |
65°C |
| VspI |
D |
<10% |
25-50% |
75-100% |
100% |
<10% |
37°C |
| XbaI |
D |
50-75% |
75-100% |
75-100% |
100% |
100% |
37°C |
| XhoI |
D |
25-50% |
75-100% |
75-100% |
100% |
10-25% |
37°C |
| XhoII |
C |
25-50% |
25-50% |
100% |
10-25% |
<10% |
37°C |
| XmaI |
B |
50-75% |
100% |
25-50% |
<10% |
50-75% |
37°C |
| XmnI |
B |
75-100% |
100% |
75-100% |
10-25% |
75-100% |
37°C |
*Sac II exhibits 100% activity with linear substrates in buffer A. It exhibits very poor activity with supercoiled substrates in this buffer.
**These buffers are not recommended due to star activity.
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Activity of Cloning Enzymes in Buffer H
EcoR I and PstI are commonly used enzymes in double digests and cloning
applications. Their optimal buffer is H. Table 3.2 provides the relative activity of other
cloning enzymes in Buffer H for double digest purposes.
|
AatII
|
<10% |
|
MluI |
100-125%* |
| AccI |
<10% |
|
NcoI |
100-125%* |
| Acc65I |
100-125%* |
|
NheI |
10-25% |
| ApaI |
<10% |
|
NotI |
100-125%* |
| AvaI |
10-25% |
|
NsiI |
>125%* |
| BamHI |
50-75% |
|
PstI |
100% |
| BbuI |
10-25% |
|
SacI |
25-50% |
| Bcl I |
50-75% |
|
SacII |
>125%* |
| Bgl I |
75-100% |
|
Sal I |
25-50% |
| BstXI |
75-100% |
|
SfiI |
50-75% |
| BstZI |
75-100% |
|
SmaI |
<10% |
| ClaI |
50-75% |
|
SpeI |
25-50% |
| CspI |
100-125%* |
|
SphI |
>125%* |
| Csp45I |
25-50% |
|
SpoI |
<10% |
| Eco52I |
50-75% |
|
SspI |
100-125%* |
| EcoRI |
100% |
|
StyI |
50-75% |
| EcoRV |
50-75% |
|
XbaI |
100-125%* |
| HincII |
50-75% |
|
XhoI |
100-125%* |
| HindIII |
25-50% |
|
XmaI |
<10% |
| KpnI |
<10% |
|
|
|
*Unit activity is based
on recommended buffer. In buffer H, some enzymes have enhanced activity.
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Composition of Promega Restriction Enzyme Reaction Buffers 1X
Promega Restriction Enzyme Reaction Buffers are provided as 10X stock
solutions. Table
3.3 provides compositions for Restriction Enzyme Reaction Buffers, listed as 1X
concentrations.
| Buffer |
pH
(at 37°C) |
Tris-HCl
(mM) |
MgCl2
(mM) |
NaCl
(mM) |
KCl
(mM) |
DTT
(mM) |
| A |
7.5 |
6 |
6 |
6 |
–
|
1 |
| B |
7.5 |
6 |
6 |
50 |
–
|
1 |
| C |
7.9 |
10 |
10 |
50 |
–
|
1 |
| D |
7.9 |
6 |
6 |
150 |
–
|
1 |
| E |
7.5 |
6 |
6 |
100 |
–
|
1 |
| F |
8.5 |
10 |
10 |
100 |
–
|
1 |
| G |
8.2 |
50 |
5 |
–
|
–
|
–
|
| H |
7.5 |
90 |
10 |
50 |
–
|
–
|
| J |
7.5 |
10 |
7 |
–
|
50 |
1 |
| K |
7.4 |
10 |
10 |
–
|
150 |
–
|
| L |
9.0 |
10 |
3 |
100 |
–
|
–
|
MULTI-CORE™ Buffer (1X) = 25mM Tris-Acetate, pH 7.5 (at 37°C), 100mM potassium
acetate, 10mM magnesium acetate, 1mM DTT.
Notes:
- For each 10°C rise in temperature between 0°C and
25°C, the pH of Tris buffers decreases 0.31 pH units.
- For each 10°C rise in temperature between 25°C and
37°C, the pH of Tris buffers decreases 0.25 pH units.
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Heat Inactivation of Restriction Enzymes
Heat-inactivation of restriction enzymes may be performed when a subsequent reaction
can be performed in the same reaction buffer, or when the reaction will be diluted for the
next application. This will eliminate the need for extra ethanol precipitations or
clean-up steps. Table 3.4 lists the sensitivity of Promega's restriction enzymes to
heat-inactivation.
Promega
Enzyme |
Heat
Inactivated |
| AatII |
+ |
| AccI |
- |
| AccIII |
- |
| Acc65I |
+ |
| AccB7I |
+ |
| AgeI |
+ |
| AluI |
+ |
| Alw26I |
+ |
| Alw44I |
+ |
| ApaI |
+ |
| AvaI |
+/- |
| AvaII |
+ |
| Bal I |
+ |
| BamHI |
+ |
| BanI |
- |
| BanII |
+ |
| BbuI |
+ |
| Bcl I |
- |
| Bgl I |
+ |
| Bgl II |
- |
| BsaMI |
- |
| BsaOI |
- |
| Bsp1286I |
+ |
| BsrBRI |
- |
| BsrS I |
- |
| BssHII |
- |
| Bst71I |
- |
| Bst98I |
- |
| BstEII |
- |
| BstOI |
- |
| BstXI |
+/- |
| BstZI |
- |
| Bsu36I |
- |
| CfoI |
+/- |
| ClaI |
+ |
| CspI |
+ |
| Csp45I |
+ |
| DdeI |
+/- |
| DpnI |
+ |
| DraI |
+ |
| EclHKI |
+ |
| Eco47III |
+ |
| Eco52I |
+ |
| EcoICRI |
+ |
| EcoRI |
+ |
| EcoRV |
+ |
| FokI |
+ |
| HaeII |
- |
| HaeIII |
- |
| HhaI |
+ |
| HincII |
+ |
| HindIII |
+ |
|
|
Promega
Enzyme |
Heat
Inactivated |
| HinfI |
- |
| HpaI |
- |
| HpaII |
- |
| Hsp92I |
+ |
| Hsp92II |
+ |
| I-Ppo I |
+ |
| KpnI |
+/- |
| MboI |
+ |
| MboII |
+ |
| MluI |
+/- |
| MspI |
+ |
| MspA1 I |
+ |
| NaeI |
+ |
| NarI |
+ |
| NciI |
+ |
| NcoI |
+ |
| NdeI |
+ |
| NdeII |
+ |
| NgoMIIV |
+ |
| NheI |
+ |
| NotI |
+ |
| NruI |
+ |
| NsiI |
+/- |
| PstI |
+ |
| PvuI |
- |
| PvuII |
+ |
| RsaI |
+ |
| SacI |
+ |
| SacII |
+ |
| Sal I |
+ |
| Sau3AI |
+ |
| Sau96I |
- |
| ScaI |
+ |
| SfiI |
- |
| SgfI(a) |
+/- |
| SinI |
+ |
| SmaI |
+ |
| SnaBI |
- |
| SpeI |
+ |
| SphI |
+ |
| SspI |
+ |
| StuI |
+ |
| StyI |
+ |
| TaqI |
- |
| Tru9I |
- |
| Tth111 I |
- |
| VspI |
+ |
| XbaI |
- |
| XhoI |
+ |
| XhoII |
+ |
| XmaI |
+ |
| XmnI |
+ |
|
Key:
+ greater than 95% inactivation (DNA is undigested).
– less that 95% inactivation (DNA digest is complete,
i.e., at least 5% of the initial 20 activity units [at least 1 unit] remain(s).)
+/– partial inactivation (DNA is partially digested).
Conditions: Twenty units of enzyme in 50µl of its optimal buffer were heated at 65°C
for 15 minutes. 1µg of DNA was added and incubated for 1 hour in accordance with the unit
definition, then analyzed by agarose gel electrophoresis.
(a)U.S. Pat. No. 5,391,487 has been issued
to Promega Corporation for Restriction Endonuclease
SgfI.
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The Effect of Site-Specific Methylation on Promega Restriction Enzymes
Heat-inactivation of restriction enzymes may be performed when a subsequent reaction can be performed in the same reaction buffer, or when the reaction will be diluted for the next application. This will eliminate the need for extra ethanol precipitations or clean-up steps. Table 3.4 lists the sensitivity of Promega's restriction enzymes to heat-inactivation.
If little or no cutting is seen with a restriction enzyme, one possibility is that DNA methylation (or lack of methylation, in the case of Dpn I) is a problem. The sensitivity of Promega's restriction enzymes to DNA methylation is summarized in Table 3.5. If the enzyme used is sensitive to methylation, check the genetic characteristics of the bacterial strain or expression system from which the DNA was purified. The interfering type of methylation may be present.
Prokaryotic Methylation
-
dcm Cytosine methylase - Methylates the C5 position of the internal cytosine residue in the sequence 5´...CCTGG...3´ and 5´...CCAGG...3´
-
dam Adenine methylase - Methylates the N6 position of the adenine residue in the sequence 5´...GATC...3´
Eukaryotic Methylation
- CpG Methylates the C5 position of the cytosine residue in the dinucleotide recognition sequence 5´...CG...3´
- CpNpGp Methylates the C5 postion of the cytosine residue in the trinucleotide 5´...CNG...3´ (N = any base)
| Enzyme |
Recognition Sequence |
dam |
dcm |
CpG |
CpNpG |
| AatII |
GACGTC |
i |
i |
s |
i |
| AccB7I |
CCANNNNNTGG |
i |
s(ol) |
i |
i |
| AccIII |
TCCGGA |
s(ol) |
i |
i |
i |
| Acc65I |
GGTACC |
i |
s(ol) |
i |
i |
| ApaI |
GGGCCC |
i |
s(ol) |
s(ol) |
i |
| AvaI |
CYCGRG |
i |
i |
s |
i |
| AvaII |
GGWCC |
i |
s(ol) |
s(ol) |
s(ol) |
| Bal I |
TGGCCA |
i |
s(ol) |
i |
s(ol) |
| BamHI |
GGATCC |
i |
i |
i |
s(ol) |
| BanII |
GRGCYC |
i |
i |
i |
i |
| BbuI |
GCATGC |
i |
i |
i |
i |
| Bcl I |
TGATCA |
s |
i |
i |
i |
| BglI |
GCCNNNNNGGC |
i |
i |
s(ol) |
s(ol) |
| BssHII |
GCGCGC |
i |
i |
s |
i |
| BglII |
AGATCT |
i |
i |
i |
s(ol) |
| BsaOI |
CGRYCG |
i |
i |
n/a |
n/a |
| Bsp1286 I |
GDGCHC |
i |
i |
i |
i |
| BsrBRI |
GATNN/NNATC |
s |
i |
i |
i |
| BstEII |
GGTNACC |
i |
i |
i |
i |
| BstOI |
CCWGG |
i |
i |
i |
n/a |
| BstXI |
CCANNNNNNTGG |
i |
i |
i |
i |
| BstZI |
CGGCCG |
i |
i |
s(ol) |
s(ol) |
| CfoI |
GCGC |
i |
i |
s |
s(ol) |
| ClaI |
ATCGAT |
s(ol) |
i |
s |
i |
| CspI |
CGGWCCG |
i |
i |
s |
s |
| Csp45I |
TTCGAA |
i |
i |
s |
i |
| DdeI |
CTNAG |
i |
i |
i |
s(ol) |
| Eco47III |
AGCGCT |
i |
i |
s |
i |
| Eco52I |
CGGCCG |
i |
i |
s |
i |
| EcoRI |
GAATTC |
i |
i |
s(ol) |
i |
| FokI |
GGATG |
i |
i |
i |
i |
| HaeIII |
GGCC |
i |
i |
i |
s(ol) |
| HhaI |
GCGC |
i |
i |
s |
s(ol) |
| HincII |
GTYRAC |
i |
i |
i |
i |
| HindIII |
AAGCTT |
i |
i |
i |
i |
| HpaII |
CCGG |
i |
i |
s |
s |
| KpnI |
GGTACC |
i |
i |
i |
i |
| MboI |
GATC |
s |
i |
i |
i |
| MboII |
GAAGA(8/7) |
s(ol) |
i |
i |
i |
| MluI |
ACGCGT |
i |
i |
s |
i |
| MspI |
CCGG |
i |
i |
s |
s |
| NaeI |
GCCGGC |
i |
i |
s |
s |
| NarI |
GGCGCC |
i |
i |
s |
i |
| NdeII |
GATC |
s |
i |
i |
i |
| NgoMIV |
GCCGGC |
i |
i |
s |
s |
| NheI |
GCTAGC |
i |
i |
s(ol) |
s(ol) |
| NotI |
GCGGCCGC |
i |
i |
s |
s |
| NruI |
TCGCGA |
s(ol) |
i |
s |
i |
| PstI |
CTGCAG |
i |
i |
i |
s |
| PvuI |
CGATCG |
i |
i |
s |
s(ol) |
| PvuII |
CAGCTG |
i |
i |
i |
s |
| SacI |
GAGCTC |
i |
i |
i |
n/a |
| SacII |
CCGCGG |
i |
i |
s |
s |
| Sal I |
GTCGAC |
i |
i |
s |
i |
| Sau3AI |
GATC |
i |
i |
s(ol) |
s(ol) |
| Sau96I |
GGNCC |
i |
s(ol) |
s(ol) |
s(ol) |
| ScaI |
AGTACT |
i |
i |
i |
i |
| SfiI |
GGCCNNNNNGGCC |
i |
s(ol) |
s(ol) |
s(ol) |
| SgfI (a) |
GCGATCGC |
i |
n/a |
s |
n/a |
| SinI |
GGWCC |
i |
i |
i |
s(ol) |
| SmaI |
CCCGGG |
i |
i |
s |
s |
| SnaBI |
TACGTA |
i |
i |
s |
i |
| SphI |
GCATGC |
i |
i |
i |
i |
| StuI |
AGGCCT |
i |
s(ol) |
i |
s(ol) |
| TaqI |
TCGA |
s(ol) |
i |
i |
i |
| Tth111 I |
GACNNNGTC |
i |
i |
i |
i |
| XbaI |
TCTAGA |
s(ol) |
i |
i |
i |
| XhoI |
CTCGAG |
i |
i |
s |
i |
| XhoII |
RGATCY |
i |
i |
i |
s(ol) |
| XmaI |
CCCGGG |
i |
i |
i |
n/a |
| XmnI |
GAANNNNTTC |
i |
i |
n/a |
n/a |
Key:
s = sensitive to this methylation
i = insensitive to this methylation
s(ol) = overlapping - (sensitive when restriction site overlaps methylation sequence)
n/a = information not available
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Methylation Sensitivity of Isoschizomer Neoschizomer Pairs
Heat-inactivation of restriction enzymes may be performed when a subsequent reaction can be performed in the same reaction buffer, or when the reaction will be diluted for the next application. This will eliminate the need for extra ethanol precipitations or clean-up steps. Table 3.4 lists the sensitivity of Promega restriction enzymes to heat-inactivation.
Isoschizomers and neoschizomers often differ in their sensitivity to methylation. This can be a simple way to differentiate the methylation state of DNA. Iso/neoschizomer pairs sold by Promega are listed below.
| Methylated Sequence |
Cleaved by |
Not Cleaved by |
| m4CCGG |
MspI (C/CGG) |
HpaII (C/CGG) |
| Cm5CGG |
MspI (C/CGG) |
HpaII (C/CGG) |
| Cm4CGG |
MspI (C/CGG) |
HpaII (C/CGG) |
| CCm5CGGG |
XmaI (C/CCGGG) |
SmaI (CCC/GGG) |
| Gm6ATC |
Sau3AI (/GATC) |
MboI, NdeII (/GATC) |
| GATm5C |
MboI, NdeII (/GATC) |
Sau3AI (/GATC) |
| GATm4C |
MboI (/GATC) |
Sau3AI (/GATC) |
| GGTACm5C |
KpnI (GGTAC/C) |
Acc65I (G/GTACC) |
Reference
McClelland, M., Nelson, M. and Raschke, E. (1994) Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucl. Acids Res. 22, 3640.
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Recognition Sites in Common DNA Substrates
Industry standards for enzyme unit definitions generally use DNA substrates other than
plasmid DNA. It is therefore necessary to determine how many units of enzyme will be
needed in cloning experiments involving plasmid vectors. Linear DNA such as lambda can be
cleaved more efficiently than closed circular plasmid DNA, and fewer units of enzyme per
microgram of DNA may be required for a complete digest. The number of sites per microgram
of DNA must also be considered. For example, 1µg of lambda contains 0.0317 picomoles of
DNA. For a 3,000bp plasmid there are 0.5pmol DNA in 1µg. Therefore, in 1µg of lambda DNA
there will be fewer cut sites to digest than in 1µg of plasmid DNA. Table 3.7 provides
information on the number of recognition sites for Promega's restriction enzymes in
various DNA substrates.
| Enzyme |
Recognition
Site |
Number of Sites in: |
| lambda |
Ad2 |
phiX174
|
pUC18
|
M13mp18
|
pBR322
|
| AatII |
GACGT/C
|
10 |
3 |
1 |
1 |
0 |
1 |
|
AccI |
GT/MKAC
|
9 |
17 |
2 |
1 |
1 |
2 |
| AccIII |
T/CCGGA
|
24 |
8 |
0 |
0 |
0 |
1 |
| Acc65I |
G/GTACC
|
2 |
8 |
0 |
1 |
1 |
0 |
| AccB7I |
CCANNNN/NTGG
|
14 |
18 |
2 |
0 |
0 |
2 |
| AgeI |
A/CCGGT
|
13 |
5 |
0 |
0 |
0 |
0 |
| AluI |
AG/CT
|
143 |
158 |
24 |
16 |
27 |
17 |
| Alw26I |
GTCTC(1/5)
|
37 |
60 |
4 |
4 |
5 |
3 |
| Alw44I |
G/TGCAC
|
4 |
7 |
1 |
3 |
0 |
3 |
| ApaI |
GGGCC/C
|
1 |
12 |
0 |
0 |
0 |
0 |
| AvaI |
C/YCGRG
|
8 |
40 |
1 |
1 |
2 |
1 |
| AvaII |
G/GWCC
|
35 |
73 |
1 |
2 |
1 |
8 |
| Bal I |
TGG/CCA
|
18 |
17 |
0 |
0 |
1 |
1 |
| BamHI |
G/GATCC
|
5 |
3 |
0 |
1 |
1 |
1 |
| BanI |
G/GYRCC
|
25 |
57 |
3 |
4 |
7 |
9 |
| BanII |
GRGCY/C
|
7 |
57 |
0 |
1 |
2 |
2 |
| BbuI |
GCATG/C
|
6 |
8 |
0 |
1 |
1 |
1 |
| Bcl I |
T/GATCA
|
8 |
5 |
0 |
0 |
0 |
0 |
| Bgl I |
GCCNNNN/NGGC
|
29 |
20 |
0 |
2 |
1 |
3 |
| Bgl II |
A/GATCT
|
6 |
11 |
0 |
0 |
1 |
0 |
| BsaMI |
GATTGCN/
|
46 |
10 |
4 |
0 |
1 |
1 |
| BsaOI |
CGRY/CG
|
22 |
50 |
1 |
5 |
4 |
7 |
| Bsp1286I |
GDGCH/C
|
38 |
105 |
3 |
10 |
5 |
10 |
| BsrBRI |
GATNN/NNATC
|
21 |
2 |
2 |
0 |
2 |
1 |
| BsrSI |
ACTGGN/
|
110 |
86 |
9 |
11 |
18 |
19 |
| BssHII |
G/CGCGC
|
6 |
52 |
1 |
0 |
0 |
0 |
| Bst71I |
GCAGC(8/12)
|
199 |
179 |
14 |
12 |
10 |
21 |
| Bst98I |
C/TTAAG
|
3 |
4 |
2 |
0 |
0 |
0 |
| BstEII |
G/GTNACC
|
13 |
10 |
0 |
0 |
0 |
0 |
| BstOI |
CC/WGG
|
71 |
136 |
2 |
5 |
7 |
6 |
| BstXI |
CCANNNNN/NTGG
|
13 |
10 |
3 |
0 |
0 |
0 |
| BstZI |
C/GGCCG
|
2 |
19 |
0 |
0 |
0 |
1 |
| Bsu36I |
CC/TNAGG
|
2 |
7 |
0 |
0 |
1 |
0 |
| CfoI |
GCG/C
|
215 |
375 |
18 |
17 |
26 |
31 |
| ClaI |
AT/CGAT
|
15 |
2 |
0 |
0 |
2
|
1
|
| CspI |
CG/GWCCG
|
5 |
2 |
0 |
0 |
0 |
0 |
| Csp45I |
TT/CGAA
|
7 |
1 |
0 |
0 |
0 |
0 |
| DdeI |
C/TNAG
|
104 |
97 |
14 |
6 |
29 |
8 |
| DpnI |
GmeA/TC
|
116 |
87 |
0 |
15 |
7 |
22 |
| DraI |
TTT/AAA
|
13 |
12 |
2 |
3 |
5 |
3 |
| EclHKI |
GACNNN/NNGTC
|
9 |
9 |
1 |
1 |
0 |
1 |
| Eco47 III |
ACG/GCT
|
2 |
13 |
0 |
0 |
2 |
4 |
| Eco52 I |
C/GGCCG
|
2 |
19 |
0 |
0 |
0 |
1 |
| EcoICRI |
GAG/CTC
|
2 |
16 |
0 |
1 |
1 |
0 |
| EcoRI |
G/AATTC
|
5 |
5 |
0 |
1 |
1 |
1 |
| EcoRV |
GAT/ATC
|
21 |
9 |
0 |
0 |
0 |
1 |
| FokI |
GGATG(9/13)
|
149 |
78 |
8 |
5 |
4 |
12 |
| HaeII |
RGCGC/Y
|
48 |
76 |
8 |
3 |
6 |
11 |
| HaeIII |
GG/CC
|
149 |
216 |
11 |
11 |
15 |
22 |
| HhaI |
GCG/C
|
215 |
375 |
18 |
17 |
26 |
31 |
| HincII |
GTY/RAC
|
35 |
25 |
13 |
1 |
1 |
2 |
| HindIII |
A/AGCTT
|
7 |
12 |
0 |
1 |
1 |
1 |
| HinfI |
G/ANTC
|
148 |
72 |
21 |
6 |
27 |
10 |
| HpaI |
GTT/AAC
|
14 |
6 |
3 |
0 |
0 |
0 |
| HpaII |
C/CGG
|
328 |
171 |
5 |
13 |
18 |
26 |
| Hsp92I |
GR/YCGC
|
40 |
44 |
7 |
3 |
1 |
6 |
| Hsp92II |
CATG/
|
181 |
183 |
22 |
11 |
16 |
26 |
| I-PpoI |
CTCTCTTAA/GGTAGC
|
0 |
0 |
0 |
0 |
0 |
0 |
| KpnI |
GGTAC/C
|
2 |
8 |
0 |
1 |
1 |
0 |
| MboI |
/GATC
|
116 |
87 |
0 |
15 |
7 |
22 |
| MboII |
GAAGA(8/7)
|
130 |
113 |
11 |
7 |
12 |
11 |
| MluI |
M/CGCGT
|
7 |
5 |
2 |
0 |
0 |
0 |
| MspI |
C/CGG
|
328 |
171 |
5 |
13 |
18 |
26 |
| MspAI |
CMG/CKG
|
75 |
95 |
4 |
6 |
4 |
5 |
| NaeI |
GCC/GGC
|
1 |
13 |
0 |
0 |
1 |
4 |
| NarI |
GG/CGCC
|
1 |
20 |
2 |
1 |
1 |
4 |
| NciI |
CC/SGG
|
114 |
97 |
1 |
7 |
4 |
10 |
| NcoI |
C/CATGG
|
4 |
20 |
0 |
0 |
0 |
0 |
| NdeI |
CA/TATG
|
7 |
2 |
0 |
1 |
3 |
1 |
| NdeII |
/GATC
|
116 |
87 |
0 |
15 |
7 |
22 |
| NgoMIV |
G/CCGGC
|
1 |
13 |
0 |
0 |
1 |
4 |
| NheI |
G/CTAGC
|
1 |
4 |
0 |
0 |
0 |
1 |
| NotI |
GC/GGCCGC
|
0 |
7 |
0 |
0 |
0 |
0 |
| NruI |
TCG/CGA
|
5 |
5 |
2 |
0 |
0 |
1 |
| NsiI |
ATGCA/T
|
14 |
9 |
0 |
0 |
0 |
0 |
| PstI |
CTGCA/G
|
28 |
30 |
1 |
1 |
1 |
1 |
| PvuI |
CGAT/CG
|
3 |
7 |
0 |
2 |
1 |
1 |
| PvuII |
CAG/CTG
|
15 |
24 |
0 |
2 |
3 |
1 |
| RsaI |
GT/AC
|
113 |
83 |
11 |
3 |
19 |
3 |
| SacI |
GAGCT/C
|
2 |
16 |
0 |
1 |
1 |
0 |
| SacII |
CCGC/GG
|
4 |
33 |
1 |
0 |
0 |
0 |
| Sal I |
G/TCGAC
|
2 |
3 |
0 |
1 |
1 |
1 |
| Sau3AI |
G/ATC
|
116 |
87 |
0 |
15 |
7 |
22 |
| Sau96I |
G/GNCC
|
74 |
164 |
2 |
6 |
4 |
15 |
| ScaI |
AGT/ACT
|
5 |
5 |
0 |
1 |
0 |
1 |
| SfiI |
GGCCNNNN/NGGCC
|
0 |
3 |
0 |
0 |
0 |
0 |
| SgfI(a) |
GCGAT/CGC
|
0 |
1 |
0 |
0 |
0 |
0 |
| SinI |
G/GWCC
|
35 |
73 |
1 |
2 |
1 |
8 |
| SmaI |
CCC/GGG
|
3 |
12 |
0 |
1 |
1 |
0 |
| SnaBI |
TAC/GTA
|
1 |
0 |
0 |
0 |
1 |
0 |
| SpeI |
A/CTAGT
|
0 |
3 |
0 |
0 |
0 |
0 |
| SphI |
GCATG/C
|
6 |
8 |
0 |
1 |
1 |
1 |
| SspI |
AAT/ATT
|
20 |
5 |
1 |
1 |
6 |
1 |
| StuI |
AGG/CCT
|
6 |
11 |
1 |
0 |
0 |
0 |
| StyI |
C/CWWGG
|
10 |
44 |
0 |
0 |
0 |
1 |
| TaqI |
T/CGA
|
121 |
50 |
10 |
4 |
13 |
7 |
| Tru9I |
T/TAA
|
195 |
115 |
35 |
13 |
62 |
15 |
| Tth111 I |
GACN/NNGTC
|
2 |
12 |
0 |
0 |
0 |
1 |
| VspI |
AT/TAAT
|
17 |
3 |
2 |
3 |
6 |
1 |
| XbaI |
T/CTAGA
|
1 |
5 |
0 |
1 |
1 |
0 |
|
XhoI |
C/TCGAG
|
1 |
6 |
1 |
0 |
0 |
0 |
| XhoII |
R/GATCY
|
21 |
22 |
0 |
7 |
3 |
8 |
| XmaI |
C/CCGGG
|
3 |
12 |
0 |
1 |
1 |
0 |
| XmnI |
GAANN/NNTTC
|
24 |
5 |
3 |
1 |
2 |
2 |
(a)U.S. Pat. No. 5,391,487 has been
issued to Promega Corporation for restriction endonuclease Sgf I.
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Multiple Cloning Sites of Promega Vectors
Promega's pGEM® Vectors and pSP Vectors are high copy number plasmid cloning vectors suitable for a wide variety of applications including routine cloning, sequencing, in vitro transcription and production of single-stranded DNA. Each vector contains a multiple cloning site featuring a variety of unique restriction enzyme sites. The multiple cloning sites of all these vectors except for the pSP64 Poly(A) Vector are flanked by SP6 and T7 phage RNA polymerase promoters. (The pSP64 Poly(A) Vector contains only the a SP6 phage promoter.) The opposing SP6 and T7 promoters allow in vitro transcription of either strand of an insert cloned into the multiple cloning site. In the pGEM® Vectors the multiple cloning site is embedded in a gene encoding the lacZ alpha-peptide, allowing selection of recombinants by blue/white color screening. The pGEM®-Zf Vectors also contain the origin of replication of the filamentous phage f1, a feature that allows synthesis of single-stranded DNA. See our vector page for more information on all of the plasmid vectors available from Promega.
|
Cloning Vector |
|
pGEM®
3Z |
pGEM®
4Z |
pGEM®
3Zf
(+/-) |
pGEM®
5Zf
(+/-) |
pGEM®
7Zf
(+/-) |
pGEM®
9Zf
(-) |
pGEM®
11Zf
(+/-) |
pGEM®
13Zf
(+) |
pSP72 pSP73 |
pSP64
Poly(A) |
| AatII |
|
|
|
x |
x |
|
|
|
|
|
| AccI |
x |
x |
x |
|
|
|
|
|
x |
x |
| Acc65I |
x |
x |
x |
|
x |
|
|
|
x |
|
| ApaI |
|
|
|
x |
x |
|
x |
|
|
|
| AvaI |
x |
x |
x |
|
|
|
|
|
|
x |
| BamHI |
x |
x |
x |
|
x |
|
x |
|
x |
x |
| Bgl II |
|
|
|
|
|
|
|
|
x |
|
| BstXI |
|
|
|
x |
x |
|
|
|
|
|
| BstZI |
|
|
|
x |
|
x |
x |
x |
|
|
| ClaI |
|
|
|
|
x |
|
|
|
x |
|
| Csp45I |
|
|
|
|
x |
|
|
|
|
|
| Eco |
|
|
|
x |
|
x |
x |
x |
|
|
| EcoICRI |
x |
x |
x |
x |
x |
x |
x |
|
x |
x |
| EcoRI |
x |
x |
x |
|
x |
x |
x |
|
x |
x |
| EcoRV |
|
|
|
x |
|
|
|
|
x |
|
| HincII |
x |
x |
x |
|
|
|
|
|
|
x |
| HindIII |
x |
x |
x |
|
x |
x |
x |
x |
x |
x |
| KpnI |
x |
x |
x |
|
x |
|
|
|
x |
|
| NcoI |
|
|
|
x |
|
|
|
|
|
|
| NdeI |
|
|
|
x |
|
|
|
|
|
|
| NotI |
|
|
|
x |
|
x |
x |
x |
|
|
| NsiI |
|
|
|
x |
x |
x |
x |
|
|
|
| PstI |
x |
x |
x |
x |
|
|
|
|
x |
x |
| PvuII |
|
|
|
|
|
|
|
|
x |
|
| SacI |
x |
x |
x |
x |
x |
x |
x |
|
x |
x |
| SacII |
|
|
|
x |
|
|
|
|
|
|
| SalI |
x |
x |
x |
x |
|
x |
x |
|
x |
x |
| SfiI |
|
|
|
|
|
x |
x |
x |
|
|
| SmaI |
x |
x |
x |
|
x |
|
|
|
x |
x |
| SpeI |
|
|
|
x |
|
x |
|
|
|
|
| SphI |
x |
x |
x |
x |
x |
|
|
|
x |
|
| StyI |
|
|
|
x |
|
|
|
|
|
|
| Tth111 I |
|
|
|
|
|
x |
|
|
|
|
| XbaI |
x |
x |
x |
|
x |
x |
x |
|
x |
x |
| XhoI |
|
|
|
|
x |
|
x |
|
x |
|
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Restriction Enzyme Recognition Sequences Containing Start and Stop Codons
Table 3.9A lists enzymes whose recognition sequences contain the start codon AUG. Those enzymes having a limited degree of degeneracy for the AUG codon (e.g., R, Y, W, etc.) are listed, however those that would require the AUG to be composed completely of unspecified nucleotides (e.g., NNN) are not included. These enzymes can be useful for creating promoter fusions in gene expression studies.
| Table 3.9A. Enzymes that have the Start Codon (AUG) as Part of Their Recognition Sequence. |
| AflIII |
A/CRYGT |
BstF5I |
GGATG(2/0) |
NlaIII |
CATG/ |
| BsaMI |
GAATGC(1/-1) |
DsaI |
C/CRYGG |
NsiI |
ATGCA/T |
| BseGI |
GGATG(2/0) |
FokI |
GGATG(9/13) |
NspI |
RCATG/Y |
| Bsp19I |
C/CATGG |
MslI |
CAYNN/NNRTG |
Ppu10I |
A/TGCAT |
| BspHI |
T/CATGA |
NcoI |
C/CATGG |
SphI |
GCATG/C |
| BspLU11 I |
A/CATGT |
NdeI |
CA/TATG |
StyI |
C/CWWGG |
| BsrDI |
GCAATG(2/0) |
|
|
|
|
Table 3.9B lists enzymes whose recognition sequences contain the stop codons UAA, UAG and UGA. Those enzymes having a limited degree of degeneracy for the stop codons (e.g. R, Y, W, etc.) are listed, however those that would require the stop codons to be composed completely of unspecified nucleotides (e.g., NNN) are not included.
| Table 3.9B. Enzymes that Have a Stop Codon (UAA, UAG, UGA) as Part of Their Recognition Sequence. |
| UAA |
UAG |
UGA |
| AseI |
AT/TAAT |
AccI |
GT/MKAC |
AsuHPI |
GGTGA(8/7) |
| Bst98I |
C/TTAAG |
AvrII |
C/CTAGG |
Bcl I |
T/GATCA |
| BstHPI |
GTT/AAC |
BfaI |
C/TAG |
BspHI |
T/CATGA |
| DraI |
TTT/AAA |
BfmI |
C/TRYAG |
Eco57I |
CTGAAG(16/14) |
| HincII |
GTY/RAC |
NheI |
G/CTAGC |
HincII |
GTY/RAC |
| HpaI |
GTT/AAC |
SfcI |
C/TRYAG |
HphI |
GGTGA(8/7) |
| PacI |
TTAAT/TAA |
SpeI |
A/CTAGT |
SmlI |
C/TYRAG |
| PmeI |
GTTT/AAAC |
StyI |
C/CWWGG |
Tsp45I |
/GTSAG |
| SmlI |
C/TYRAC |
XbaI |
T/CTAGA |
|
|
| SwaI |
ATTT/AAAT |
|
|
|
|
| Tru9I |
T/TAA |
|
|
|
|
| VspI |
AT/TAAT |
|
|
|
|
Back to top
IUPAC Ambiguity Codes for Nucleotide Degeneracy
| R = A or G |
K = G or T |
S = G or C |
| Y = C or T |
M = A or C |
W = A or T |
| B = not A (C, G or T) |
H = not G (A, C or T) |
N = any nucleotide |
| D = not C (A, G or T) |
V = not T (A, C or G) |
|
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