Technical Appendix

Transcription

Technical Appendix

Technical Appendix
Restriction Enzyme Activity in Promega 10X Buffers, Reaction Temperature and Heat Inactivation.
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
Promega’s 4-CORE® 10X Buffers. Many commonly used cloning enzymes (i.e. restriction sites are found in vector multiple cloning sites) have buffers E and
H as their optimal buffer and so we have determined the activity of many of our other restriction enzymes in those buffers as well. This table may be used
to select the best buffer for digestion with multiple restriction enzymes. Enzyme activity is expressed as a percent of the activity obtained with the optimal
buffer for each enzyme in a one-hour digest. Enzymes with 100% activity (green) perform as well as the optimal buffer. Enzymes with 50–75% or 75–100%
(yellow) will give acceptable activity in that buffer. Enzymes with <10%, 10–25%, or 25–50% (pink) activity generally do not have acceptable activity in that
buffer. Also, buffers leading to star activity of the enzyme (*; pink) should be avoided. If compatible buffers cannot be identified with acceptable activity for
both enzymes, each digest should be performed separately in the optimal buffer for each enzyme.
Promega
Enzyme
Aat II
Acc I
Acc III
Acc 65 I
Acc B7 I
Age I
Alu I
Alw 26 I
Alw 44 I
Apa I
Ava I
Ava II
Bal I
Bam H I
Ban I
Ban II
Bbu I
Bcl I
Bgl I
Bgl II
Bsa M I
Bsp 1286 I
Bsr S I
Bss H II
Bst 98 I
Bst E II
Bst O I
Bst X I
Bst Z I
Bsu 36 I
Cfo I
Cla I
Csp I
Csp 45 I
Dde I
Dpn I
Dra I
Ecl HK I
Eco 47 III
Eco 52 I
Eco ICR I
Eco R I
Eco R V
Fok I
Hae II
Hae III
Hha I
Hin c II
Hin d III
Hin f I
Cat.#
Buffer
Supplied
with Enzyme
Activity in
A
B
R6541
R6411
R6581
R6921
R7081
R7251
R6281
R6761
R6771
R6361
R6091
R6131
R6691
R6021
R6891
R6561
R6621
R6651
R6071
R6081
R6991
R6741
R7241
R6831
R7141
R6641
R6931
R6471
R6881
R6821
R6241
R6551
R6671
R6571
R6291
R6231
R6271
R7111
R6731
R6751
R6951
R6011
R6351
R6781
R6661
R6171
R6441
R6031
R6041
R6201
J
G
F
D
E
K
B
C
C
A
B
C
G
E
G
E
A
C
D
D
D
A
D
H
D
D
C
D
D
E
B
C
K
B
D
B
B
E
D
L
B
H
D
B
B
C
C
B
E
B
50–75%
50–75%
<10%
10–25%
10–25%
25–50%
75–100%
10–25%
<10%
100%
10–25%
50–75%
10–25%
75–100%*
25–50%
75–100%
100%
10–25%
10–25%
25–50%
10–25%
100%
10–25%
75–100%
<10%
25–50%
10–25%
<10%
<10%
<10%
75–100%
75–100%
<10%
25–50%
25–50%
50–75%
75–100%
<10%
<10%
<10%
10–25%
25–50%
10–25%
75–100%
50–75%
75–100%
50–75%
25–50%
25–50%
50–75%
10–25%
25–50%
10–25%
50–75%
50–75%
25–50%
100%
25–50%
25–50%
50–75%
100%
50–75%
<10%
75–100%
25–50%
75–100%
75–100%
75–100%
25–50%
75–100%
25–50%
50–75%
25–50%
50–75%
10–25%
50–75%
25–50%
10–25%
<10%
25–50%
100%
75–100%
10–25%
100%
25–50%
100%
100%
<10%
25–50%
<10%
100%
50–75%
25–50%
100%
100%
75–100%
75–100%
100%
100%
100%
C
D
<10%
<10%
25–50% 10–25%
25–50% 25–50%
75–100%
100%
100%*
<10%
25–50% 50–75%
75–100% 10–25%
100%
10–25%
100%
25–50%
50–75%
<10%
50–75% 25–50%
100%
25–50%
<10%
<10%
75–100% 50–75%
10–25%
<10%
75–100% 25–50%
75–100%
<10%
100%
50–75%
75–100%
100%
75–100%
100%
50–75%
100%
25–50% 10–25%
10–25%
100%
75–100% 50–75%
10–25%
100%
50–75%
100%
100%
25–50%
25–50%
100%
10–25%
100%
50–75% 25–50%
75–100% 25–50%
100%
75–100%
25–50% 50–75%
50–75% 25–50%
50–75%
100%
75–100% 50–75%
75–100% 50–75%
75–100% 10–25%
50–75%
100%
10–25% 25–50%
75–100%
<10%
50–75% 50–75%
50–75%
100%
75–100% 25–50%
50–75% 10–25%
100%
50–75%
100%
50–75%
25–50% 50–75%
75–100% 10–25%
75–100% 75–100%
www.promega.com • [email protected]
E
H
MULTICORE™
10–25%
<10%
<10%
<10%
<10%
25–50%
n.d.
n.d.
<10%
75–100% 100–125%** 100%
100%
n.d.
100%
n.d.
n.d.
100%
n.d.
n.d.
10–25%
n.d.
n.d.
75–100%
n.d.
n.d.
100%
10–25%
<10%
75–100%
100%
10–25%
<10%
n.d.
n.d.
25–50%
n.d.
n.d.
<10%
100%
50–75% 75–100%
n.d.
n.d.
100%
n.d.
n.d.
100%
10–25% 10–25%
100%
50–75% 50–75% 10–25%
25–50% 75–100%
100%
n.d.
n.d.
<10%
n.d.
n.d.
25–50%
n.d.
n.d.
75–100%
n.d.
n.d.
100%
n.d.
100%
75–100%
n.d.
n.d.
25–50%
n.d.
n.d.
100%
n.d.
n.d.
<10%
100%
75–100% 10–25%
10–25% 75–100% 10–25%
100%
n.d.
50–75%
n.d.
n.d.
100%
100%
50–75%
100%
100% 100–125%** 10–25%
100%
25–50% 50–75%
n.d.
n.d.
25–50%
n.d.
n.d.
100%
n.d.
n.d.
25–50%
100%
n.d.
50–75%
n.d.
n.d.
25–50%
25–50% 50–75%
<10%
25–50%
n.d.
100%
75–100%
100%
100%*
25–50% 50–75%
100%
n.d.
n.d.
50–75%
n.d.
n.d.
100%
n.d.
n.d.
100%
n.d.
n.d.
75–100%
75–100% 50–75%
100%
100%
25–50% 50–75%
n.d.
n.d.
50–75%
Enzyme
Heat
Assay
Inactivation Temperature
+
–
–
+
+
+
+
+
+
+
+/–
+
+
+
–
+
+
–
+
–
–
+
–
–
–
–
–
+/–
–
–
+/–
+
+
+
+/–
+
+
+
+
+
+
+
+
+
–
–
+
+
+
–
37°C
37°C
65°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
50°C
37°C
37°C
50°C
37°C
37°C
65°C
37°C
65°C
50°C
37°C
60°C
60°C
50°C
50°C
37°C
37°C
37°C
30°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
57
Technical Appendix
Restriction Enzyme Activity in Promega 10X Buffers, Reaction Temperature and Heat Inactivation (continued).
58
Promega
Enzyme
Hpa I
Hpa II
Hsp 92 I
Hsp 92 II
I-Ppo I
Kpn I
Mbo I
Mbo II
Mlu I
Msp I
Msp A1 I
Nae I
Nar I
Nci I
Nco I
Nde I
Nde II
Ngo M IV
Nhe I
Not I
Nru I
Nsi I
Pst I
Pvu I
Pvu II
Rsa I
Sac I
Sac II
Sal I
Sau 3A I
Sca I
Sfi I
Sgf I
Sin I
Sma I
Sna B I
Spe I
Sph I
Ssp I
Stu I
Sty I
Taq I
Tru 9 I
Tth 111 I
Vsp I
Xba I
Xho I
Xho II
Xma I
Xmn I
Cat.#
Buffer
Supplied
with Enzyme
Activity in
A
B
C
D
E
H
R6301
R6311
R7151
R7161
R7031
R6341
R6711
R6723
R6381
R6401
R7021
R7131
R6861
R7061
R6513
R6801
R7291
R7171
R6501
R6431
R7091
R6531
R6111
R6321
R6331
R6371
R6061
R6221
R6051
R6191
R6211
R6391
R7103
R6141
R6121
R6791
R6591
R6261
R6601
R6421
R6481
R6151
R7011
R6841
R6851
R6181
R6161
R6811
R6491
R7271
J
A
F
K
NA
J
C
B
D
B
C
A
G
B
D
D
D
MULTI-CORE™
B
D
K
D
H
D
B
C
J
C
D
B
K
B
C
A
J
B
B
K
E
B
F
E
F
B
D
D
D
C
B
B
25–50%
100%
10–25%
10–25%
10–25%
100%*
10–25%
10–25%
10–25%
75–100%
25–50%
100%
75–100%
100%*
50–75%
<10%
<10%
100%*
75–100%
<10%
<10%
10–25%
10–25%
10–25%
25–50%
75–100%
75–100%
100%
<10%
25–50%
<10%
75–100%
25–50%
100%
<10%
50–75%
75–100%
75–100%
10–25%
75–100%
25–50%
10–25%
75–100%
50–75%
<10%
50–75%
25–50%
25–50%
50–75%
75–100%
50–75%
50–75%
75–100%
25–50%
25–50%
25–50%
75–100%
100%
25–50%
100%
100%*
50–75%
50–75%
100%
75–100%
<10%
<10%
100%*
100%
10–25%
<10%
50–75%
50–75%
25–50%
100%
75–100%
25–50%
50–75%
10–25%
100%
100%*
100%
25–50%
75–100%
<10%
100%
100%
75–100%
50–75%
100%
75–100%
25–50%
50–75%
100%
25–50%
75–100%
75–100%
25–50%
100%
100%
25–50%
50–75%
50–75%
25–50%
25–50%
25–50%
100%
50–75%
50–75%
75–100%
100%
25–50%
75–100%
25–50%
75–100%
25–50%
10–25%
100%*
75–100%
25–50%
<10%
50–75%
50–75%
50–75%
50–75%
100%
25–50%
100%
25–50%
75–100%
50–75%
75–100%
100%
50–75%
<10%
50–75%
75–100%
100%*
50–75%
75–100%
75–100%
50–75%
75–100%
75–100%
75–100%
75–100%
75–100%
100%
25–50%
75–100%
10–25%
10–25%
25–50%
<10%
25–50%
<10%
50–75%
75–100%
100%
25–50%
10–25%
<10%
25–50%
25–50%
100%
100%
100%
<10%
10–25%
100%
50–75%
100%
50–75%
100%
25–50%
<10%
<10%
50–75%
100%
<10%
75–100%
25–50%
<10%
10–25%
<10%
<10%
75–100%
75–100%
75–100%
50–75%
75–100%
50–75%
25–50%
25–50%
100%
100%
100%
10–25%
<10%
10–25%
n.d.
n.d.
n.d.
n.d.
n.d.
25–50%
n.d.
n.d.
25–50%
n.d.
n.d.
n.d.
n.d.
n.d.
100%
n.d.
n.d.
n.d.
75–100%
25–50%
n.d.
25–50%
25–50%
n.d.
n.d.
n.d.
100%
25–50%
25–50%
n.d.
n.d.
75–100%
n.d.
n.d.
<10%
n.d.
100%
100%
100%
n.d.
10–25%
100%
n.d.
n.d.
n.d.
100%
25–50%
n.d.
25–50%
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
<10%
n.d.
n.d.
100–125%**
n.d.
n.d.
n.d.
n.d.
n.d.
100–125%**
n.d.
n.d.
n.d.
10–25%
100–125%**
n.d.
>125%**
100%
n.d.
n.d.
n.d.
25–50%
>125%**
25–50%
n.d.
n.d.
50–75%
n.d.
n.d.
<10%
n.d.
25–50%
>125%**
100–125%**
n.d.
50–75%
n.d.
n.d.
n.d.
n.d.
100–125%**
100–125%**
n.d.
<10%
n.d.
MULTICORE™
100%
100%
10–25%
<10%
—
75–100%
<10%
100%
10–25%
25–50%
100%
50–75%
50–75%
50–75%
75–100%
25–50%
25–50%
100%
100%
25–50%
10–25%
10–25%
25–50%
<10%
50–75%
<10%
100%
<10%
<10%
100%
10–25%
75–100%
<10%
100%
100%
100%
100%
10–25%
50–75%
50–75%
<10%
100%
25–50%
100%
<10%
100%
10–25%
<10%
50–75%
75–100%
Enzyme
Heat
Assay
Inactivation Temperature
–
–
+
+
+
+/–
+
+
+/–
+
+
+
+
+
+
+
+
+
+
+
+
+/–
+
–
+
+
+
+
+
+
+
–
+/–
+
+
–
+
+
+
+
+
–
–
–
+
–
+
+
+
+
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
37°C
50°C
37°C
37°C
25°C
37°C
37°C
37°C
37°C
37°C
37°C
65°C
65°C
65°C
37°C
37°C
37°C
37°C
37°C
37°C
* Not recommended due to potential star activity.
** Unit activity is based on recommended buffer. In Buffer H, some enzymes have enhanced activity.
n.d. = Not determined.
Heat Inactivation Key:
+
=greater than 95% inactivation (DNA is undigested)
–
=less than 95% inactivation (DNA digest is complete, i.e., ≥5% of the initial 20 activity units [≥1 unit] remains)
+/–
=partial inactivation (DNA is partially digested)
Heat Inactivation Conditions:
Twenty units of enzyme in 50µl of its optimal buffer were heated at 65°C for 15 minutes.
One microgram of DNA was added and incubated for 1 hour in accordance with the unit definition, then analyzed by agarose gel electrophoresis.
Promega Subcloning Notebook
Technical Appendix
Isoschizomers. The enzymes in boldface type are available from Promega.
Enzyme
mAat I
Aat II
Acc I
Acc III
Acc 65 I
Isoschizomer(s)
Stu I, Eco 147 I, Pme 55 I, Sse B I
—
Fbl I, Xmi I
Bsp E II, Mro I
Asp718 I
Kpn I*
Acc B1 I
Ban I, Bsh N I, Eco 64 I
Acc B7 I Pfl M I, Van 91 I
Acl N I
Spe l
Acl W I
Alw I
Acy I
Bbi II, Hin 1 I, Hsp 92 I,
Bsa H I, Msp 171 I
Acs I
Apo I
Afa I
Csp 6 I*, Rsa I
Afe I
Eco 47 III
Afl II
Bst 98 I
Age I
Pin A I
Aha III
Dra I
Ahd I
Ecl HK I
Alu I
—
Alw I
Acl W I
Alw 26 I 1 Bsm A I
Alw 44 I Apa L I
Aoc I
Bsu 36 I, Cvn I
Apa I
Bsp 120 I
Apa L I
Alw 44 I, Vne I
Apo I
Acs I
Ase I
Vsp I, Asn I
Asn I
Vsp I, Ase I
Asp I
Tth 111 I
Asp E I
Ahd I, Eam 1105 I, Ecl HK I
Asp 700 I Xmn I
Asp 718 I Acc 65 I
Kpn I*
Asu I
Sau 96 I, Cfr 13 I
Asu II
Csp 45 I, Bst B I
Asu HP I
Hph I
Ava I
Ama 87 I, Bco I, Bso B I, Eco 88 I
Ava II
Sin I, Eco 47 I, Hgi E I
Axy I
Bsu 36 I
Bal I
Msc I, Mlu N I
Bam H I —
Ban I
Acc BI, Bsh N I, Eco 64 I
Ban II
Eco 24 I
Bbe I
—
Nar I*
Bbr P I
Eco 72 I, Pml I
Bbs I1
Bsc 91 I, Bpi I
Bbu I
Pae I, Sph I
Bcl I
Bsi Q I, Fba I
Bcn I
Nci I
Bfr I
Bst 98 I
Bgl I
—
Bgl II
—
Bmy I
Bsp1286 I
Bpm I
Gsu I
Bsa H I
Hsp 92 I
Bsa M I
Bsm I
Bsa O I
Bsh 1285 I, Bsi E I
Bse A I
Acc III
Bse N I
Bsr S I, Bsr I
Bse P I
Bss H II, Pau I
Bsh 1285 I Bsa O I
Bsh N I
Ban I, Acc B1 I, Eco 64 I
Bsh 1365 I Bsr BR I
Bsi E I
Bsa O I
Recognition Sequence
AGG▼CCT
GACGT▼C
GT▼(A/C)(G/T)AC
T▼CCGGA
G▼GTACC
GGTAC▼C
G▼G(C/T)(G/A)CC
CCAN4▼NTGG
A▼CTAGT
GGATCNNNN▼
G(A/G)▼CG(T/C)C
(G/A)▼AATT(C/T)
GT▼AC
AGC▼GCT
C▼TTAAGG
A▼CCGGT
TTT▼AAA
GACNNN▼NNGTC
AG▼CT
GGATCNNNN▼
GTCTC(1/5)
G▼TGCAC
CC▼TNAGG
GGGCC▼C
G▼TGCAC
(G/A)▼AATT(C/T)
AT▼TAAT
AT▼TAAT
GACN▼NNGTC
GACNNN/NNGTC
GAANN▼NNTTC
G▼GTACC
GGTAC▼C
G▼GNCC
TT▼CGAA
GGTGAN8▼
C▼(C/T)CG(G/A)G
G▼G(A/T)CC
CC▼TNAGG
TGG▼CCA
G▼GATCC
G▼G(T/C)(A/G)CC
G(A/G)GC(T/C)▼C
GGCGC▼C
GG▼CGCC
CAC▼GTG
GAAGAC(2/6)
GCATG▼C
T▼GATCA
CC▼(C/G)GG
C▼TTAAG
GCCNNNN▼NGGC
A▼GATCT
G(G/A/T)GC(C/A/T)▼C
CTGGAG(16/14)
G(A/G)▼CG(T/C)C
GAATGC(1/–1)
CG(A/G)(T/C)▼C
T▼CCGGA
ACTGGN (1/–1)
G▼CGCGC
CG(A/G)(T/C)▼CG
G▼G(T/C)(A/G)CC
GATNN▼NNATC
CG(A/G)(T/C)▼CG
Enzyme
Bsm I
Bsm A I1
Bso B I
Bsp 19 I
Bsp 68 I
Bsp 106 I
Bsp 119 I
Bsp 120 I
Bsp 143 I
Bsp 143 II
Bsp 1286 I
Bsp C I
Bsp D I
Bsp E I
Bsr I1
Bsr S I1
Bss H II
Bst 98 I
Bst B I
Bst E II
Bst N I
Bst O I
Bst X I
Bst Y I
Bst Z I
Bsu 15 I
Bsu 36 I
Bsu R I
Cfo I
Isoschizomer(s)
Bsa MI
Alw 26 I
Ava I, Ama 87 I, Bco I, Eco 88 I
Nco I
Nru I
Cla I, Bsa D I
Csp 45 I, Nsp V, Bst B I
Apa I
Mbo I, Sau 3A I, Nde II
Hae II
Bmy I, Sdu I
Pvu I
Cla I
Acc III
Bsr S I, Bse N I
Bse N I, Bsr I
Bse P I, Pau I
Afl II, Bfr I
Csp 45 I, Nsp V, Bsp 119 I
Bst P I, Eco 91 I, Psp E I
Bst O I, Mva I, Eco R II
Bst N I, Eco R II, Mva I
–
Xho II, Mfl I
Eco 52 I, Eag I, Xma III, Ecl X I
Cla I
Cvn I, Aoc I, Eco 81 I
Hae III, Pal I
Hha I
Hin 6 I
Hin P1 I*
Cfr 9 I
Xma I
Sma I*
Cfr 13 I
Sau 96 I
Cfr 42 I
Sac II
Cla I
Ban III, Bsp 106 I, Bsp D I, Bsu 15 I
Cpo I
Csp I, Rsr II
Csp I
Cpo I, Rsr II
Csp 6 I
Rsa I*, Afa I*
Csp 45 I Bst B I, Nsp V, Bsp119 I
Cvn I
Bsu 36 I
Dde I
Bst DE I
Dpn I 2
Dpn II*
Dpn II
Mbo I, Sau 3A I, Nde II, Dpn I*
Dra I
—
Eag I
Eco 52 I, Bst Z I, Ecl X I, Xma III
Eam1105 I Ecl HK I, Ahd I, Asp E I
Ecl 136 II Eco ICR I
Sac I*
Ecl HK I Ahd I, Eam 1105 I, Asp E I
Ecl X I
Bst Z I, Eag I, Eco 52 I, Xma III
Eco 24 I
Ban II, Fri O I
Eco 32 I
Eco R V
Eco 47 I
Ava II, Sin I
Eco 47 III Afe I
Eco 52 I Bst Z I, Xma III, Eag I, Ecl X I
Eco 64 I
Ban I, Bsh N I, Eco 64 I
Eco 81 I
Bsu 36 I
Eco 88 I
Ava I
Eco 91 I
Bst E II
Eco 105 I Sna B I
Eco 130 I Sty I
Eco 147 I Stu I
Eco ICR I Ecl 136 II
Sac I*
Sst I*
GAATGCN▼
GTCTC(1/5)
C(C/T)CG(G/A)G
C▼CATGG
TCG▼CGA
AT▼CGAT
TT▼CGAA
G▼GGCCC
▼GATC
(A/G)GCGC▼(T/C)
CGAT▼CG
AT▼CGAT
T▼CCGGA
ACTGGN(1/-1)
ACTGGN(1/-1)
G▼CGCGC
C▼TTAAG
TT▼CGAA
G▼GTNACC
CC▼(A/T)GG
CC▼(A/T)GG
CCANNNNN▼NTGG
(A/G)▼GATC(T/C)
C▼GGCCG
AT▼CGAT
CC▼TNAGG
GG▼CC
GCG▼C
GCG▼C
G▼CGC
C▼CCGGG
CCC▼GGG
G▼GNCC
CCGC▼GG
AT▼CGAT
CG▼G(A/T)CCG
CG▼G(A/T)CCG
GT▼AC
TT▼CGAA
CC▼TNAGG
C▼TNAG
GmeA▼TC
▼GATC
TTT▼AAA
C▼GGCCG
GACNNN▼NNGTC
GAG▼CTC
GAGCT▼C
GACNNN▼NNGTC
C▼GGCCG
G(AG)GC(TC)▼C
GAT▼ATC
G▼G(A/T)CC
AGC▼GCT
C▼GGCCG
G▼G(TC)(AG)CC
CC▼TNAGG
C▼(TC)CG(AG)G
G▼GTNACC
TAC▼CTA
C▼C(A/T)(T/A)GG
AGG▼CCT
GAG▼CTC
GAGCT▼C
GAGCT▼C
59
Technical Appendix
Isoschizomers (continued).
Enzyme
Eco R I
Eco R II
Eco R V
Eco T14 I
Eco T22 I
Ehe I
Fok I 2
Hae II
Hae III
Hap II
Hgi E I
Hha I
Hin 1 I
Hin c II
Hin d II
Hin d III
Hin f I
Hin P1 I
Hpa I
Hpa II3
Hsp 92 I
Hsp 92 II
I-Ppo I
Kas I
Kpn I
Ksp I
Mbo I
Mbo II1
Mfl I
Mlu I
Mlu N I
Mro I
Msc I
Mse I
Msp I 3
Msp A1 I
Mst II
Mva I
Nae I
Nar I
Nci I
Nco I
Nde I
Nde II
NgoM IV
Nhe I
Nla III
Not I
Nru I
Nsi I
Nsp V
Nsp B II
Pae I
Pae R7 I
Pal I
Pfl M I
Pin A I
Pst I
Pvu I
Pvu II
60
The enzymes in boldface type are available from Promega.
Isoschizomer(s)
—
Bst O I, Bst N I, Mva I
Eco 32 I
Sty I
Nsi I
Nar I*
—
Bsp 143 II
Bsu R I, Pal I
Hpa II, Msp I
Eco 47 I, Sin I, Ava II
Cfo I
Hin P1 I*, Hin 6 I*
Acy I, Hsp 92 I
Hin d II
Hin c II
—
—
—
Hha I*, Cfo I*
Ksp A I
Msp I, Hap II
Acy I, Bsa H I, Hin 1 I
Nla III
—
Nar I*
—
Acc 65 I*, Asp 718 I*
Sac II
Sau 3A I, Nde II, Dpn II
—
Xho II
—
Bal I, Msc I
Acc III
Bal I, Mlu N I
Tru 9 I
Hpa II, Hap II
Nsp B II
Bsu 36 I
Bst O I, Eco R II, Bst N I
Ngo M IV
—
Ehe I*
Kas I*
Bbe I*
Bcn I
Bsp 19 I
—
Mbo I, Sau 3A I, Dpn II
Nae I
—
Hsp 92 II
—
Bsp 68 I
Eco T22 I, Mph 1103 I
Csp 45 I, Bst B I, Bsp 119 I
Msp A1 I
Bbu I, Sph I
Xho I
Hae III, Bsu R I
Acc B7 I, Vau 91 I
Age I
—
Bsp C I
—
G▼AATTC
CC▼(A/T)GG
GAT▼ATC
C▼C(A/T)(A/T)GG
ATGCA▼T
GG▼CGCC
GGATG(9/13)
(A/G)GCGC▼(T/C)
GG▼CC
C▼CGG
G▼G(A/T)CC
GCG▼C
G▼CGC
G(A/G)▼CG(T/C)C
GT(T/C)▼(A/G)AC
GT(T/C)▼(A/G)AC
A▼AGCTT
G▼ANTC
G▼CGC
GCG▼C
GTT▼AAC
C▼CGG
G(A/G)▼CG(C/T)
CATG▼
CTCTCTTAA▼GGTAGC
GG▼CGCC
GGTAC▼C
G▼GTACC
CCGC▼GG
▼GATC
GAAGA(8/7)
(A/G)▼GATC(T/C)
A▼CGCGT
TGG▼CCA
T▼CCGGA
TGG▼CCA
T▼TAA
C▼CGG
C(A/C)G▼C(G/T)G
CC▼TNAGG
CC▼(A/T)GG
G▼CCGGC
GG▼CGCC
GGC▼GCC
G▼GCGCC
GGCGC▼C
CC▼(C/G)GG
C▼CATGG
CA▼TATG
▼GATC
G▼CCGGC
G▼CTAGC
CATG▼
GC▼GGCCGC
TCG▼CGA
ATGCA▼T
TT▼CGAA
C(A/C)G▼C(G/T)G
GCATG▼C
C▼TCGAG
GG▼CC
CCAN4▼NTGG
A▼CCGGT
CTGCA▼G
CGAT▼CG
CAG▼CTG
Enzyme
Rsa I
Rsr II
Sac I
Sac II
Sal I
Sau 3A I
Sau 96 I
Sca I
Sdu I
Sfi I
Sfu I
Sgf I
Sin I
Sma I
Sna B I
Spe I
Sph I
Ssp I
Sst I
Sst II
Stu I
Sty I
Taq I
Tru 9 I
Tth 111 I
Tth HB8 I
Van 91 I
Vne I
Vsp I
Xba I
Xho I
Xho II
Xma I
Xma III
Xma C I
Xmn I
Isoschizomer(s)
Afa I
Csp I, Cpo I
Sst I
Ecl 136 II*, Eco ICR I*
Sst II, Ksp I, Cfr 42 I
—
Mbo I, Nde II, Dpn II
Cfr 13 I
—
Bsp 1286 I
—
Csp 45 I
—
Ava II, Eco 47 I
—
Xma I*, Cfr 9 I*
Eco 105 I
Acl N I
Bbu I, Pae I
—
Sac I
Eco ICR I*
Sac II
Aat I, Eco 147 I
Eco T14 I
Tth HB8 I
Mse I
Asp I
Taq I
Acc B7 I, Pfl M I
Apa L I, Alw 44 I
Ase I, Asn I
—
Pae R7 I
Bst Y I, Mfl I
Cfr 9 I, Xma C I,
Sma I*
Eco 52 I, Bst Z I, Eag I, Ecl X I
Xma I
Sma I*
Asp 700 I
GT▼AC
CG▼G(A/T)CCG
GAGCT▼C
GAG▼CTC
CCGC▼GG
G▼TCGAC
▼GATC
G▼GNCC
AGT▼ACT
GGCCNNNN▼NGGCC
TT▼CGAA
GCGAT▼CGC
G▼G(A/T)CC
CCC▼GGG
C▼CCGGG
TAC▼GTA
A▼CTAGT
GCATG▼C
AAT▼ATT
GAGCT▼C
GAG▼CTC
CCGC▼GG
AGG▼CCT
C▼C(A/T)(A/T)GG
T▼CGA
T▼TAA
GACN▼NNGTC
T▼CGA
CCAN4▼NTGG
G▼TGCAC
AT▼TAAT
T▼CTAGA
C▼TCGAG
(A/G)▼GATC(T/C)
C▼CCGGG
CCC▼GGG
C▼GGCCG
C▼CCGGG
CCC▼GGG
GAANN▼NNTTC
Key:
N = A, C, G or T
* = neoschizomer
Notes:
1. The locations of cleavage sites falling outside the recognition site are indicated in
parentheses. For example, GTCTC(1/5) indicates cleavage at:
5′...GTCTCN▼...3′
3′...CAGAGNNNNN▲...5′
2. Dpn I is unique among commercially available restriction enzymes in requiring
methylation of a nucleotide (adenine) in its recognition sequence in order to cut.
Therefore, Dpn I cannot be substituted for other enzymes recognizing the GATC sequence
(e.g., Mbo I and Sau 3A I).
3. Although Hpa II and Msp I recognize the same nucleotide sequence, Hpa II is sensitive
to methylation of either cytosine in its recognition sequence, while Msp I is sensitive
only to methylation of the external cytosine. These enzymes may not be interchanged for
all applications.
Reference
Roberts, R.J. (1991) Nucl. Acids Res. 19 (supp), 2077–109.
Technical Appendix
Compatible Ends.
Promega Restriction Enzymes That Generate 5′ Overhangs
Promega Restriction Enzymes That Generate 3′ Overhangs
Overhang
5′-N
5′-S
5′-W
5′-AT
5′-CG
Overhang
N-3′
AT-3′
CG-3′
CN-3′
GC-3′
NNN-3′
ACGT-3′
AGCT-3′
CATG-3′
DGCH-3′
GCGC-3′
GGCC-3′
GTAC-3′
NNNN-3′
RGCY-3′
TGCA-3′
TTAA-3′
5′-GN
5′-MK
5′-TA
5′-ANT
5′-GNC
5′-GWC
5′-TNA
5′-AATT
5′-AGCT
5′-CATG
5′-CCGG
5′-CGCG
5′-CTAG
5′-CWWG
5′-GATC
5′-GCGC
5′-GGCC
5′-GTAC
5′-GTNAC
5′-GYRC
5′-TCGA
5′-TGCA
5′-TTAA
5′-YCGR
Definite Compatible Ends
Possible Compatible Ends
Tth 111 I
Nci I
Bst O I
Acc I
Nar I, Msp I, Hsp 92 I,
Taq I, Cla I, Csp 45 I, Hpa II
Bsr S I
Acc I
Vsp I, Nde I, Tru 9 I
Hin f I
Sau 96 I
Ava II, Csp I, Sin I
Dde I, Bsu 36 I
Eco R I
Hin d III
Nco I
Age I, Xma I, Acc III,
Ngo M IV
Mlu I, Bss H I
Spe I, Nhe I, Xba I
Mbo I, Sau 3A I, Bam H I,
Bgl II, Xho II, Bcl I, Nde II
Ban I
Not I, Bst Z I, Eco 52 I
Acc 65 I
Sal I, Xho I
Alw 44 I
Bst 98 I
Sty I
Ava I
Sty I
Sty I
Definite Compatible Ends
Possible Compatible Ends
Ecl HK I
Sgf I, Pvu I
Cfo I, Hha I
Sac II
Bsa M I
Bsa O I
Acc B7 I, Bgl I, Sfi I
Aat II
Sac I
Hsp 92 II, Sph I, Bbu I
Ban II, Bsp 1286 I
Bsp 1286 I
Hae II
Apa I
Kpn I
Nsi I, Pst I
I-Ppo I
Ban II, Bsp 1286 I
Bst X I
Ban II, Bsp 1286 I
Bsp1286 I
Key:
D
H
K
M
N
R
S
W
Y
=
=
=
=
=
=
=
=
=
A or G or T
A or C or T
G or T
A or C
A or C or G or T
A or G
C or G
A or T
C or T
Ban I
Bst E II
Ban I
Ava I
Ava I
61
Technical Appendix
Site-Specific Methylation Sensitivity of Promega Restriction Enzymes.
This table lists the sensitivities of several Promega restriction enzymes to site-specific methylation at dam, dcm, CpG and CpNpG sites (p = phosphoryl group).
These four modifications are frequently found in DNA of bacteria, eukaryotes or their viruses. Many strains of E. coli contain the site-specific dam and dcm DNA
methylases. Higher eukaryotes contain the site-specific CpG and CpNpG DNA methylases. In mammalian genomes, methylation occurs mainly at the CG
dinucleotide. In plant genomes, methylation may occur at both the CG and CNG sequences.
Prokaryotic Methylation
dcm
dam
Cytosine methylase mutation—methylates the C5 position of the
internal cytosine residue in the sequence 5′...CCTGG...3′.
Adenine methylase mutation—methylates the N6 position of the
adenine residue in the sequence 5′....GATC...3′.
For further information regarding site-specific methylation, refer to McClelland,
M., Nelson, M. and Raschke, E. (1994) Nucl. Acids Res. 22, 3640–59.
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
Eukaryotic Methylation
CpG
Methylates the C5 position of the cytosine residue in the dinucleotide
recognition sequence 5′...CG...3′.
CpNpGp Methylates the C5 position of the cytosine residue in the trinucleotide
recognition sequence 5′...CNG...3′ (N = any base).
Enzyme
Aat II
Acc B7 I
Acc III
Acc 65 I
Apa I
Ava I
Ava II
Bal I
Bam H I
Ban II
Bbu I
Bcl I
Bgl I
Bgl II
Bsp 1286 I
Bss H II
Bst E II
Bst O I
Bst X I
Bst Z I
Cfo I
Cla I
Csp I
Csp 45 I
Dde I
Eco 47 III
Eco 52 I
Eco R I
Fok I
Hae III
Hha I
Hin c II
Hin d III
Hpa II
62
GACGTC
CCANNNNNTGG
TCCGGA
GGTACC
GGGCCC
CYCGRG
GGWCC
TGGCCA
GGATCC
GRGCYC
GCATGC
TGATCA
GCCNNNNNGGC
AGATCT
GDGCHC
GCGCGC
GGTNACC
CCWGG
CCANNNNNNTGG
CGGCCG
GCGC
ATCGAT
CGGWCCG
TTCGAA
CTNAG
AGCGCT
CGGCCG
GAATTC
GGATC
GGCC
GCGC
GTYRAC
AAGCTT
CCGG
dam
i
i
s(ol)
i
i
i
i
i
i
i
i
s
i
i
i
i
i
i
i
i
i
s(ol)
i
i
i
i
i
i
i
i
i
i
i
i
dcm
i
s(ol)
i
s(ol)
s(ol)
i
s(ol)
s(ol)
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
CpG
s
i
i
i
s(ol)
s
s(ol)
i
i
i
i
i
s(ol)
i
i
s
i
i
i
s(ol)
s
s
i
s
i
s
s
s(ol)
i
i
s
i
i
s
CpNpG
i
i
i
i
i
i
s(ol)
s(ol)
s(ol)
i
i
i
s(ol)
s(ol)
i
i
i
n/a
i
s(ol)
n/a
i
s
i
s(ol)
i
i
i
i
s(ol)
s(ol)
i
i
s
Enzyme
Kpn I
Mbo II
Mlu I
Msp I
Nae I
Nar I
Nde II
Ngo M IV
Nhe I
Not I
Nru I
Pst I
Pvu I
Pvu II
Sac I
Sac II
Sal I
Sau 3A I
Sau 96 I
Sca I
Sfi I
Sgf I
Sin I
Sma I
Sna B I
Sph I
Stu I
Taq I
Xba I
Xho I
Xho II
Xma I
Xmn I
GGTACC
GAAGA(8/7)
ACGCGT
CCGG
GCCGGC
GGCGCC
GATC
GCCGGC
GCTAGC
GCGGCCGC
TCGCGA
CTGCAG
CGATCG
CAGCTG
GAGCTC
CCGCGG
GTCGAC
GATC
GGNCC
AGTACT
GGCCNNNNNGGCC
GCGATCGC
GGWCC
CCCGGG
TACGTA
GCATGC
AGGCCT
TCGA
TCTAGA
CTCGAG
RGATCY
CCCGGG
GAANNNN
dam
i
s(ol)
i
i
i
i
s
i
i
i
s(ol)
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
s(ol)
s(ol)
i
i
i
i
dcm
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
s(ol)
i
s(ol)
i
i
i
i
i
s(ol)
i
i
i
i
i
i
CpG
i
i
s
i
s
s
i
s
s(ol)
s
s
i
s
i
i
s
s
s(ol)
s(ol)
i
s(ol)
s
i
s
s
i
i
i
i
s
i
i
n/a
CpNpG
i
i
i
s
s
i
i
s
s(ol)
s
i
s
s(ol)
s
i
s
n/a
s(ol)
s(ol)
i
s(ol)
n/a
s(ol)
s
i
i
s(ol)
i
i
i
s(ol)
n/a
n/a
Technical Appendix
Restriction Enzyme Buffer Composition.
Buffer
A
B
C
D
E
F
G
H
J
K
L
pH
(at 37°C)
7.5
7.5
7.9
7.9
7.5
8.5
8.2
7.5
7.5
7.4
9.0
Tris-HCl
(mM)
6
6
10
6
6
10
50
90
10
10
10
MgCl2
(mM)
6
6
10
6
6
10
5
10
7
10
3
NaCl
(mM)
6
50
50
150
100
100
—
50
—
—
100
Copy Number of Commonly Used Plasmids.
KCl
(mM)
—
—
—
—
—
—
—
—
50
150
—
DTT
(mM)
1
1
1
1
1
1
—
—
1
—
—
MULTI-CORE™ Buffer (1X) = 25mM Tris-acetate (pH 7.5 at 37°C), 100mM potassium
acetate, 10mM magnesium acetate, 1mM DTT.
Notes:
1. For each 10°C rise in temperature between 0°C and 25°C, the pH of
Tris buffers decreases 0.31 pH units.
2. For each 10°C rise in temperature between 25°C and 37°C, the pH of
Tris buffers decreases 0.25 pH units.
3. All of Promega’s Restriction enzymes are supplied with 10mg/ml Acetylated
BSA. Although BSA is not absolutely required for activity, it has been
shown to enhance activity of many restriction enzymes. We recommend
adding BSA to all restriction digests at a final concentration of 0.1mg.ml.
Plasmid
Size
(approx.)
2,700bp
2,700bp
4,400bp
4,500bp
4,000bp
9,000bp
5,000bp
4,000bp
4,000bp
4,000bp
Plasmid
pGEM®
pUC
pBR322
ColE1
pACYC
pSC101
pGL Series
pRL Series
phRL Series
phRG Series
pGEM®-T/
T easy
psiLentGene™
Series
psiCHECK™ 1/2
psiSTRIKE™
Series
pALTER®-1/Ex 1
pALTER®-Ex 2
pSP
pCI, pSI
Origin of
Replication*
mutated pMB1
mutated pMB1
pMB1
ColE1
p15A
pSC101
mutated pMB1
mutated pMB1
mutated pMB1
mutated pMB1
Copy
Number
300–700
500–700
>25
>15
~10
~6
300–700
300–700
300–700
300–700
**Yield
per ml of
Culture Reference
1.8–4.1µg
1
2.9–4.1µg
1
>0.23µg
2
>0.15µg
3
~0.09µg
4
~0.12µg
5
3.3–7.6µg
1
2.7–6.0µg
1
2.7–6.0µg
1
2.7–6.0µg
1
3,000bp mutated pMB1 300–700 2.0–4.6µg
1
4,000bp mutated pMB1 300–700 2.7–6.0µg
3,500bp mutated pMB1 300–700 2.7–5.3µg
1
1
4,000bp mutated pMB1 300-700 2.7–6.0µg
5,800bp
pMB1
>25
>0.3µg
5,800bp
p15A
~10
~0.13µg
2,500bp mutated pMB1 300–700 1.6–3.8µg
3,600bp mutated pMB1 300–700 2.4–5.5µg
1
3
4
1
1
* Plasmids carrying the pMB1, mutated pMBI and ColE1 belong to the same
incompatibility group, so they are not compatible with one another, but they are fully
compatible with those carrying p15A and pSC101 replicons.
** Theoretical plasmid yields were calculated from the reported copy number and size of
each plasmid assuming 2.0 × 109 cells per milliliter of culture grown for 16 hours at 37°C.
References
1. Summerton, J., Atkins, T. and Bestwick, R. (1983) Anal. Biochem.
133, 79–84.
2. Holmes, D.S. and Quigley, M. (1981) Anal. Biochem. 114, 193–7.
3. Jansz, H.S., Pouwels, P.H. and Schiphorst, J. (1966) Biochem. Biophys.
Acta 123, 626.
4. Birnboim, H.C. and Doly, J. (1979) Nucl. Acids Res. 7, 1513–23.
5. Birnboim, H.C. (1983) Meth. Enzymol. 100, 243–55.
Star Activity.
Restriction enzymes, under nonstandard conditions, can demonstrate the ability to cleave DNA at sequences different from their defined recognition sites. The term
“star activity” has been given to this nonsequence-specific cleavage of DNA under nonoptimal reaction conditions. The most common types of altered activity are
single-base substitutions, truncation of the outer bases in the recognition sequence and single-strand nicking (1). In general, star activity is not a concern if
restriction endonucleases are used in the recommended buffers at the appropriate temperatures. Star activity is evident with a number of restriction enzymes when
the following parameters are altered in the reaction environment (2):
•
•
•
•
•
•
High enzyme concentration (generally >100 units/µg).
High glycerol content (>5% v/v).
Substitution of Mn2+ for Mg2+ (or substitution of other divalent cations).
Low salt concentration (generally <25mM).
Extremes of pH, especially pH>8.0.
Presence of DMSO, ethanol or other organic solvents.
References
1. Barany, F. (1988) Gene 65, 149–65.
2. Brown, T.A., Hames, B.D. and Rickwood, D. (1991) In: Molecular Biology Lab Fax, BIOS Scientific Publishers Limited, Oxford, United Kingdom.
63
Technical Appendix
Genotypes of Frequently Used Bacterial Strains.
All genes in the bacterium are presumed to be in the wildtype state, except for those listed, which are mutant alleles carried by that bacterium. Genes listed on the
F′ episome, however, represent wildtype alleles unless specified otherwise. Strains are λ– unless specified otherwise. *Strains available from Promega as competent
cells are indicated by an asterisk. Strains shown in bold are available from Promega as glycerol freezer stocks.
Strain
BL21(DE3)
*BL21(DE3)pLysS
*BMH 71-18 mut S
C600 (1)
C600hfl (1)
DH1 (2)
DH10B
DH5α™
DM1 (3)
ES1301 mut S
*HB101 (4)
JM101 (5)
*JM109 (5)
JM109(DE3) (5)
JM110 (5)
KW251
LE392 (6)
NM522 (7)
NM538 (8)
NM539 (8)
*Select96™
Stbl2™
Stbl4™
SURE®
TOP10
TOP10F′
XL1-Blue
Y1089 (9)
Y1090 (9)
Genotype
F–, omp T, hsd SB (rB–, mB–), dcm, gal, λ(DE3)
F–, omp T, hsd SB (rB–, mB–), dcm, gal, λ(DE3), pLysS (Cmr)
thi, supE, ∆(lac-proAB), [mutS ::Tn10(tetr)] [F′, tra D36, proAB, laqI q Z∆M15]
thi -1, thr -1, leuB 6, lacY 1, tonA 21, supE 44
thi -1, thr -1, leuB 6, lacY 1, tonA 21, supE 44, hfl A150::Tn10(tetr)
recA 1, endA 1, gyrA 96, thi -1, hsdR 17 (rK–, mK+), supE 44, relA 1
F–, mcr A ∆(mrr-hsd RMS-mcr BC) φ80lac Z∆M15, ∆lac X74, deo R, rec A1, end A1, ara D139, ∆(ara, leu )7697, gal U, gal K, λ-,
rps L(strr), nup G
φ 80dlacZ∆M15, recA 1, endA 1, gyrA 96, thi -1, hsdR 17 (rK–, mK+), supE 44, relA 1, deoR, ∆(lacZYA-argF ) U169, pho A
F′, dam -13::Tn9(Cmr) dcm, mcrB, hsdr-M+, gal 1, gal 2, ara-, lac -, thr -, leu -, ton R, tsx R, Su o
lacZ 53, thyA 36, rha -5, metB 1, deo C, IN(rrn D-rrn E), [mutS 201::Tn5]
thi -1, hsdS 20 (rB–, mB–), supE 44, recA 13, ara -14, leuB 6, proA 2, lacY 1, gal K2, rpsL 20(strr), xyl -5, mtl -1
supE, thi, ∆(lac-proAB ), F′ (traD 36, proAB, lacI qZ ∆M15)
endA1, recA1, gyrA 96, thi -1, hsdR 17 (rK–, mK+), relA 1, supE 44, ∆(lac-proAB ), [F′, traD 36, proAB, lacI qZ∆M15]
endA1, recA1, gyrA 96, thi -1, hsdR 17 (rK–, mK+), relA 1, supE 44, ∆(lac-proAB ), [F′, traD 36, proAB, lacI qZ∆M15], λ(DE3)
rpsL(strr), thr, leu, thi, hsdR 17 (rK–, mK+), lacY, galK, galT, ara, tonA, tsx, dam, dcm, supE 44, ∆(lac-proAB ), [F′, traD 36, proAB,
lacI qZ∆M15]
supE 44, galK 2, galT 22, metB 1, hsdR 2, mcrB 1, mcrA, [argA 81::Tn10(tetr)], rec D1014
hsdR 514, (rK–, mK+), supE 44, supF 58, lacY 1 or ∆(lacIZY )6, galK 2, galT 22, metB 1, trpR 55
supE, thi, ∆(lac-proAB ), ∆hsd 5 (rK–, mK–), [F′, proAB, lacI qZ∆M15]
supF, hsdR (rK–, m K+), trpR, lacY
supF, hsdR (rK–, m K+), lacY, (P2)
mcr A, ∆(mrr -hsd RMS-mcr BC), φ80lacZ∆M15, ∆lac X74, rec A1, ara D139 (ara -leu )7697, gal U, gal K, rsp L, end A1, nup G
F–, mcr A, ∆(mcr BC-hsd RMS-mrr ), rec A1, end A1, gyr A96, thi -1, sup E44, rel A1, λ–, ∆(lac -pro AB)
mcr A, ∆(mcr BC-hsd RMS-mrr ), rec A1, end A1, gyr A96, thi -1, sup E44, rel A1, λ–, ∆(lac -pro AB), gal, F′{pro AB+, lac lq, Z∆M15, Tn 10(tet R)}
e 14–, (mcr A–) ∆(mcr CB-hsd SMR-mrr )171, end A1, sup E44, thi -1, gyr A96, rel A1, lac, rec B, rec J, sbc C, umu C::Tn5 (kan r), uvr C,
[F′ pro AB, lac lqZ∆M15::Tn10 (tet r)]
F–, mcr A, ∆(mrr -hsd RMS-mcr BC), φ80lac Z∆M15, ∆lac X74, deo R, rec A1, ara D139, ∆(ara , leu )7697, gal U, gal K, rps L (strR), end A1,
nup G
F′{lac lq Tn10 (tetR)}, mcr A, ∆(mrr -hsd RMS-mcr BC), φ80lac Z∆M15, ∆lac X74, deo R, rec A1, ara D139, ∆(ara -leu )7697, gal U, gal K,
rps L (strr), end A1, nup G
recA 1, endA 1, gyrA 96, thi -1, hsdR 17(rK–, mK+), supE 44, relA 1, lac, [F′, proAB, lacl qZ∆M15::Tn10(tetr)]
∆(lac U169), proA +, ∆(lon ), araD 139, strA, hflA 150, [chr::Tn10(tetr)], (pMC9)
∆(lac U169), proA +, ∆(lon ), araD 139, strA, supF, rpsL(strr), [trpC 22::Tn10 (tetr)], (pMC9), hsdR (rK–, mK+)
Miscellaneous
F′ Host contains an F′ episome with the stated features.
λ(DE3) Bacteriophage λ carrying the gene for T7 RNA polymerase is integrated
into the host genome.
pMC9 is pBR322 with lac Iq inserted and confers amp and tet resistance.
64
References
1. Jendrisak, J., Young, R.A. and Engel, J. (1987) In: Guide to Molecular
Cloning Techniques, Berger, S. and Kimmel, A., eds., Academic Press, San
Diego, CA.
2. Hanahan, D. (1983) J. Mol. Biol. 166, 557–80.
3. Lorow-Murray, D. and Bloom, F. (1991) Focus 13, 20.
4. Lacks, S. and Greenberg, J.B. (1977) J. Mol. Biol. 114, 153–60.
5. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Gene 33, 103–19.
6. Murray, N. et al. (1977) Mol. Gen. Genet. 150, 53–61.
7. Gough, J. and Murray, N. (1983) J. Mol. Bio. 166, 1–19.
8. Frischauf, A. et al. (1983) J. Mol. Biol. 170, 827–42.
9. Huynh, T., Young, R.A. and Davis, R. (1985) In: DNA Cloning, Vol. 1,
Glover, D., ed., IRL Press Ltd., Oxford, UK.
Technical Appendix
Genetic Markers in E. coli.
Symbol
ara -14
ara D
arg A
cyc A
dam
dap D
dcm
deo C
deo R
dut 1
endA 1
gal E
gal K
gal T
gyrA 96
hflA 150
hfl B
hsdR
(rK–, mK+)
hsdS 20
(rB–, mB–)
lacI q
lacY
lacZ ∆M15
leuB
∆ (lon )
Lys S
mcrA
mcrB
metB
met C
Description
Mutation in arabinose metabolism
L-ribulose phosphate 4-epimerase mutation;
part of an inducible operon araBAD repressed
by L-arabinose
N-Acetylglutamate synthase mutation; inhibited
by the presence of arginine
Involved in D-alanine, glycine, D-serine and
D-cycloserine transport, and an L-alanine carrier
DNA adenine methylase mutation
Succinyl-diaminopimelate aminotransferase
mutation
DNA cytosine methylase mutation
Deoxyribose-phosphate aldolase mutation
Regulatory gene mutation allowing constitutive
expression of genes for deoxyribose synthesis
Mutation of deoxyuridine triphosphatase, which
catalyzes dUTP the conversion to dUMP and PPi
DNA-specific endonuclease I mutation
Part of the galETK operon that encodes
UDP galactose-4-epimerase
Galactokinase mutation
Galactose-1-phosphate uridylyltransferase mutation
DNA gyrase mutation
Protease mutation that leads to stabilization
of cII gene products
Gene encodes a possible protease component
Host DNA restriction and methylation system
mutation: Restriction minus, modification positive
for the E. coli K strain methylation system
Mutation of specificity determinant for host DNA
restriction and methylation system. Restriction
minus, modification minus for the E. coli B strain
methylation system
Overproduction of the lac repressor protein
Galactoside permease mutation
Partial deletion of
β-D-galactosidase gene
β-isopropylmalate dehydrogenase mutation
Deletion of lon protease
pLysS plasmid is integrated into the host genome
mtl
mutS
ompT
P2
pro A
Mutation in methylcytosine restriction system
Mutation in methylcytosine restriction system
Cystathionine γ-synthase mutation
Cystathionine beta-lyase mutation; involved in
methionine biosynthesis
Mutation in mannitol metabolism
Methyl-directed mismatch repair mutation
Mutation of protease VII, an outer membrane protein
P2 bacteriophage lysogen present in host
γ-glutamyl phoshate reductase mutation
proAB
Mutations in proline metabolism
Effect of Mutation
Blocks arabinose catabolism.
Blocks arabinose catabolism.
Arginine required from growth in minimal media.
Mutants cannot use D-alanine as a carbon source.
Blocks methylation of adenine residues in the sequence
5′…GmATC…3′.
Mutant reflects impaired synthesis of succinyl CoA and needs to be supplemented with
succinate or lysine + methionine.
Blocks methylation of cytosine in the sequence
5′…CmCAGG…3′ or 5′…CmCTGG…3′.
Allows efficient propagation of large plasmids.
Mutants are impaired in conversion of dUTP to dUMP, leading to higher dUTP pools that can
lead to misincorporation of uracil instead of thymidine. Stable incorporation of dUTP needs
mutation in ung gene.
Improves quality of plasmid DNA isolations.
Mutant is more resistant to bacteriophage P1 infection.
Blocks catabolism of galactose.
Blocks catabolism of galactose.
Confers resistance to nalidixic acid.
Leads to high frequency of lysogeny by λ phages (1).
Mutations lead to high frequency of bacteriophage lambda lysogenization.
Allows cloning without cleavage of transformed DNA by endogenous restriction endonucleases.
DNA prepared from this strain can be used to transform rK+ E. coli strains.
Allows cloning without cleavage of transformed DNA by endogenous restriction
endonucleases. DNA prepared from this strain is unmethylated by the hsdS 20 methylases.
Leads to high levels of the lac repressor protein, inhibiting transcription from the lac promoter.
Blocks lactose utilization.
Allows complementation of β-galactosidase activity by α-complementation sequence in
pGEM®-Z Vectors. Allows blue/white selection for recombinant colonies when plated on X-Gal.
Requires leucine for growth on minimal media.
Reduces proteolysis of expressed proteins.
Strains carrying this plasmid will be tet resistant and produce T7 lysozyme, a natural inhibitor
of T7 RNA polymerase, thus lowering background transcription of sequences under the
control of the T7 RNA polymerase promoter (2).
Blocks restriction of DNA methylated at the sequence 5′…GmCGC…3′.
Blocks restriction of DNA methylated at the sequence 5′…AGmCT…3′.
Requires methionine for growth on minimal media.
Methionine required from growth in minimal media.
Blocks catabolism of mannitol.
Prevents repair of the newly synthesized, unmethylated strand.
Reduces proteolysis of expressed proteins.
λ phages containing the red and gam genes of λ are growth inhibited by P2 lysogens (3).
proA/argD mutant will not block proline synthesis, but will be repressed by arginine. Mutants
excrete proline on minimal media and are resistant to proline analogs. proA/argD/argR triple
mutant grows slowly on minimal media + arginine.
Requires proline for growth in minimal media.
65
Technical Appendix
Genetic Markers in E. coli (continued).
Symbol
recA 1,
recA 13
recB, recC
rec D
rec F
rel A
rha
rpsL
sbcB
str A
supB, supC,
supG, supL,
supM, supN,
supO
supD, supE,
supF
thi -1
thr
thy A
Tn5
Tn10
tonA
traD 36
trp C
trp R
tsx
ung 1
xyl -5
Description
Mutation in recombination
Exonuclease V mutations
The Rec BCD trimer (exonuclease V) progressively
degrades ssDNA and dsDNA in an ATP-dependent
manner to form oligonucleotides; implicated in
homologous recombination
Recombination and repair mutation
ppGpp synthetase I mutation, a novel nucleotide
guanosine 5′-diphosphate-3′-diphosphate
produced in response to starvation by relA
ribosomal protein sensing uncharged tRNA
Utilization of L-rhamnose, a methylpentose
Mutation in subunit S12 of 30S ribosome
Exonuclease I mutation
Mutant alters ribosome protein S12
Suppressor mutations
Effect of Mutation
Minimizes recombination of introduced DNA with host DNA, increasing stability of
inserts. Inserts are more stable in recA 1 than recA 13 hosts.
Reduces general recombination and affects repair of radiation damage.
Allows easier propagation of sequences with inverted repeats.
Mutant cannot repair daughter strand gaps (post-replicational repair).
Allows RNA synthesis in the absence of protein synthesis.
Blocks rhamnose catabolism.
Confers resistance to streptomycin.
Allows general recombination in rec BC mutant strains.
Confers resistance to streptomycin
Suppresses ochre (UAA) and amber (UAG) mutations.
Suppressor mutations
Suppresses amber (UAG) mutations.
Mutation in thiamine metabolism
Threonine biosynthesis mutation
Thymidylate synthase; dTTP biosynthesis
Transposon
Transposon
Mutation in outer membrane protein
Transfer factor mutation
Phosphoribosyl anthranilate isomerase mutation;
part of tryptophan biosynthesis pathway
trpR aporepressor; regulates the biosynthesis
of tryptophan and its transport
T6 and colicin K phage receptor; outer membrane
protein involved in specific diffusion of nucleosides;
transports the antibotic albicidin
Uracil-DNA N-glycosylase
Mutation in xylose metabolism
Thiamine required for growth in minimal media.
Mutants are obligate threonine auxotrophs.
Mutants are obligate thymidine auxotrophs.
Encodes resistance to kanamycin.
Encodes resistance to tetracycline.
Confers resistance to bacteriophage T1.
Prevents transfer of F′ episome.
Resistant to bacteriophage T6 and colicin K.
Allows uracil to exist in plasmid DNA.
Blocks catabolism of xylose.
References
1.
2.
3.
4.
66
Hoyt, M.A. et. al. (1982) Cell 31, 565–73.
Studier, F.W. (1991) J. Mol. Biol. 219, 37–44.
Kaiser, K. and Murray, N. (1985) In: DNA Cloning, Vol. 1, Glover, D., ed., IRL Press Ltd., Oxford, UK.
Neidnardt, F. ed. (1996) Escherichia coli and Salmonella Cellular and Molecular Biology 2nd ed, ASM Press, Washington, D.C.
Technical Appendix
Nucleic Acids and Proteins: Calculations.
An online calculator for these values is available in the “tools” section of
Promega’s Web site at: www.promega.com/techserv/tools/
Formulas for DNA Molar Conversions
For dsDNA:
To convert pmol to µg:
Metric Prefixes
pmol ×
Prefix
kilo
centi
milli
micro
nano
pico
femto
atto
zepto
Symbol
Factor
k
c
m
µ
n
p
f
a
z
10 3
10–2
10–3
10–6
10–9
10–12
10–15
10–18
10–21
N
×
660pg
1µg
×
=
pmol
106pg
µg
To convert µg to pmol:
pmol
1
106pg
×
×
= pmol
µg
×
1µg
660pg
N
where N is the number of nucleotide pairs and 660pg/pmol is the average
MW of a nucleotide pair.
For ssDNA:
To convert pmol to µg:
Spectrophotometric Conversions
1 A260 unit of double-stranded DNA = 50µg/ml
1 A260 unit of single-stranded DNA = 33µg/ml
1 A260 unit of single-stranded RNA = 40µg/ml
DNA Molar Conversions
1µg of 1,000bp DNA
1µg of pBR322 DNA
1pmol of 1,000bp DNA
1pmol of pBR322 DNA
=
=
=
=
1.52pmol (3.03pmol of ends)
0.36pmol DNA
0.66µg
2.8µg
pmol ×
N
×
330pg
1µg
×
=
pmol
106pg
µg
To convert µg to pmol:
pmol
1
106pg
×
×
= pmol
µg
×
1µg
330pg
N
where N is the number of nucleotides and 330pg/pmol is the average MW of
a nucleotide.
Dalton (Da) is an alternate name for the atomic mass unit, and kiloDalton (kDa) is 1,000 Daltons.
Thus a protein with a mass of 64kDa has a molecular weight of 64,000 grams per mole.
(a)
The PCR process is covered by patents issued and applicable in certain countries*. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process.
*In the U.S., effective March 29, 2005, U.S. Pat. Nos. 4,683,195, 4,965,188 and 4,683,202 will expire. In Europe, effective March 28, 2006, European Pat. Nos. 201,184 and 200,362 will expire.
(b)
Purchase of this product is accompanied by a limited license under U.S. Pat. Nos. 5,082,784 and 5,192,675 for the internal research use of the computer.
(c)
Turbo™ Nae I and Turbo™ Nar I are the subjects of U.S. Pat. Nos. 5,248,600 and 5,418,150 and are licensed exclusively to Promega Corporation, as well as a license under DD 264 231.
(d)
Licensed using U.S. Pat. No. 4,935,361.
(e)
Licensed under U.S. Pat. No. 5,7075.
(f)
Licensed using one or more of U.S. Pat. Nos. 5,487,993 and 5,827,657 and European Pat. No. 0 550 693.
(g)
U.S. Pat. No. 4,766,072.
(h)
The PCR process, which is the subject of European Pat. Nos. 201,184 and 200,362 and U.S. Pat. Nos. 4,683,195, 4,965,188 and 4,683,202 owned by Hoffmann-LaRoche*, is covered by patents issued and applicable
in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this product is recommended for persons that either have a license to perform PCR or are
not required to obtain a license.
*In the U.S., effective March 29, 2005, the above primary U.S. Pat. Nos. 4,683,195, 4,965,188 and 4,683,202 will expire. In Europe, effective March 28, 2006, the above primary European Pat. Nos. 201,184 and
200,362 will expire.
(i)
Certain applications of this product are covered by patents issued and applicable in certain countries. Because purchase of this product does not include a license to perform any patented application, users of this
product may be required to obtain a patent license depending upon the particular application and country in which the product is used.
(j)
Australian Pat. No. 730718 and other patents and patents pending.
(k)
U.S. Pat. No. 5,981,235, Australian Pat. No. 729932 and other patents pending.
©2004 Promega Corporation. All Rights Reserved.
All prices and specifications are subject to change without prior notice. Product claims are subject to change. Please contact Promega Technical Services or access Promega online for the most up-to-date information on
Promega products.
GeneEditor, LigaFast, MULTICORE, Select96, TaqBead, and Turbo are trademarks of Promega Corporation. 4-CORE, Altered Sites, GoTaq, pALTER, pGEM, VacMan and Wizard are trademarks of Promega Corporation and
are registered with the U.S. Patent and Trademark Office.
ABLE and SURE are registered trademarks of Stratagene. Bacto is a registered trademark of Difco Laboratories, Detroit, Michigan. DH5α is a trademark of Life Technologies, Inc. Ficoll is a registered trademark of
Amersham Biosciences Ltd. Stb12 adn Stb14 are trademarks of Invitrogen Corporation.
67

Technical Appendix

Transcription

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