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%
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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
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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*
Recognition Sequence 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) G(G/A/T)GC(C/A/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 —
Recognition Sequence 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
Recognition Sequence GT▼AC CG▼G(A/T)CCG GAGCT▼C GAG▼CTC CCGC▼GG G▼TCGAC ▼GATC G▼GNCC AGT▼ACT G(G/A/T)GC(C/A/T)▼C 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.
Promega Subcloning Notebook
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
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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
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Recognition Sequence 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
Recognition Sequence 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
Promega Subcloning Notebook
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.
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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.
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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.
Promega Subcloning Notebook
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.
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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.
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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.
Promega Subcloning Notebook
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.
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