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1、窗体顶端Select 1st enzyme: Select 2nd enzyme: 窗体底端EnzymeCat#TempSupplied NEBufferSupplements% Activity in NEBufferBSASAM1234BamHI*R013637CNEBuffer 3YesNo75100100100KpnI*R014237CNEBuffer 1YesNo10075050Double Digest Recommendation(s) for BamHI + KpnI: Double digest is not recommended. A sequential digest
2、is required, using each enzyme in its supplied NEBuffer.Note: The above recommendation is based on the experimental results. Please check Suggested NEBuffers for Double Digestion.*BamHI has a High Fidelity version BamHI-HF (R3136)KpnI has a High Fidelity version KpnI-HF (R3142)High Fidelity (HF) Res
3、triction Enzymes have been engineered for reduced star activityand have 100% activity in buffer 4 which may simplify your double digest.Double DigestsDigesting a DNA substrate with two restriction endonucleases simultaneously (double digestion) is a common timesaving procedure. Selecting the best NE
4、Buffer to provide reaction conditions that optimize enzyme activity as well as avoid star activity associated with some enzymes is an important consideration. Each enzyme is supplied with its optimal NEBuffer to ensure 100% activity. NEBuffer compositions are listed on buffer pages and on the data c
5、ard sent with each enzyme. The Activity Chart for Restriction Enzymes rates the percentage activity of each restriction endonuclease in the four standard NEBuffers and NEBuffer EcoRI.Please use NEB Double Digest Finder to perform a double digest calculation.Suggested NEBuffers for Double DigestionEn
6、zymeAatIIAvrIIBamHIBglIIBsgIEagIEcoRIEcoRVHindIIIKpnIMseINcoINdeINheINotIPstIPvuISacISacIISalISmaISpeISphIXbaIXhoIXmaINEBuffer443343EcoRI3214342333143442444AatII4-4seqseq4seqseq4444444seq4seq44seq444444AvrII44-3243EcoRI2214442332143442444BamHI3seq3-333EcoRI3seqseq333seq333seqseq3seqseq333seqBglII3se
7、q23-33EcoRI3222332333223seq22232BsgI44433-seqseq42seq4444333443444444EagI3seq333seq-EcoRI3seqseq333seq333seqseq3seqseq333seqEcoRIEcoRIseqEcoRIEcoRIEcoRIseqEcoRI-EcoRIseq1EcoRIEcoRIEcoRI1EcoRIEcoRIEcoRI1EcoRIEcoRIseqEcoRIEcoRIseqEcoRIseqEcoRV3423343EcoRI-222322333223422234HindIII242seq22seqseq2-22222
8、22222seq42222seqKpnI141seq2seqseq122-111121214seqseq11214MseI4443243EcoRI221-442233443442444NcoI3443343EcoRI3214-42333143442444NdeI4443343EcoRI22144-4333443442444NheI242seq24seq1221224-22214seq422224NotI3seq33333EcoRI3222332-33223seq22332PstI3433333EcoRI32133323-3123422334PvuI3seq23333EcoRI322333233
9、-223seq22332SacI141seq24seq12214141212-4seq411414SacII444seq24seqEcoRI22444442224-seq444444SalI3seq33333EcoRI3seqseq333seq333seqseq-seqseq333seqSmaI444seqseq4seqseq44seq4444seq4seq44seq-44444SpeI444seq24seqEcoRI221444222214seq4-2444SphI2423243EcoRI221222222214342-224XbaI4443243seq2224442333443442-44
10、XhoI4443343EcoRI32144423331434424-4XmaI444seq24seqseq4seq4444424244seq44444-Note: Enzymes in Dark Red are available in High Fidelity (HF) Format. HF Enzymes have been engineered for reduced star activity and have 100% activity in NEBuffer 4 which may simplify your double digest. Click for more HF in
11、formation.Setting up a Double Digestion Choose an NEBuffer that results in the most activity for both enzymes. If star activity is a concern, consider using one of our High Fidelity (HF) enzymes. If BSA is required for either enzyme, add it to the double digest reaction (BSA does not inhibit any res
12、triction enzyme). Set up reaction according to recommended conditions. The final concentration of glycerol in any reaction should be less than 5% to minimize the possibility of star activity. For example, in a 50 l reaction, the total amount of enzyme added should not exceed 5 l. Incubate at recomme
13、nded temperature. Overnight double digests should be avoided due to the possibility of star activity. If two different incubation temperatures are necessary, choose the optimal reaction buffer and set up reaction accordingly. Add the first enzyme and incubate at the desired temperature, Then, heat k
14、ill the first enzyme, add the second enzyme and incubate at the recommended temperature. Depending on an enzymes activity rating in a non-optimal NEBuffer, the number of units or incubation time may be adjusted to compensate for the slower rate of cleavage. Setting up a Double Digestion with a Uniqu
15、e Buffer (designated “U”) Our buffer system has been streamlined, leaving three enzymes that have unique buffers: EcoRI (included in this chart and the Restriction Enzyme Activity Chart), SspI (same buffer composition as EcoRI) and DpnII. In most cases, DpnII requires a sequential digest. Setting up
16、 a Sequential Digestion Set up a reaction using the restriction endonuclease that has the lowest salt concentration in its recommended buffer and incubate to completion. Adjust the salt concentration of the reaction (using a small volume of a concentrated salt solution) to approximate the reaction c
17、onditions of the second restriction endonuclease. Add the second enzyme and incubate to complete the second reaction. Alternatively, a spin column can be used to isolate the DNA prior to the second reaction. Double Digest FAQ Spotlight:Q: I would like to digest DNA with EcoRI and XbaI at the same ti
18、me. The Double Digest Finder recommends a sequential digest, using each enzyme in its supplied NEBuffer. However, both of these enzymes show 100% activity in NEBuffer 2. Why is a double digest not recommended with these enzymes?A: Although EcoRI shows 100% activity in NEBuffer 2, it also exhibits si
19、gnificant star activity in this buffer, resulting in non-specific, unintended cleavage. This is also observed when using NEBuffer 4. For this reason, a sequential（相继的） digest is recommended. However, research has shown that EcoRI exhibits less star activity in NEBuffers 1 and 3. Since XbaI has 75% a
20、ctivity in NEBuffer 3 a double digest could be performed in this buffer, provided that the reactions are set up according to the recommended reaction conditions for avoiding star activity.In this case, EcoRI is available in High Fedelity (HF) Format. HF Enzymes have been engineered for reduced star
21、activity and have 100% activity in NEBuffer 4 which may simplify your double digest.Double Digest Finder窗体顶端Cleaving a DNA substrate with two restriction enzymes simultaneously (double digestion) is a common timesaving procedure. Use this tool to select reaction conditions amenable to any two NEB re
22、striction enzymes.Click here for more information on double digests and a chart of double digests for some common enzyme combinations. Select 1st enzyme: Select 2nd enzyme: 窗体底端EnzymeCat#TempSupplied NEBufferSupplements% Activity in NEBufferBSASAM1234BamHI-HFR313637CNEBuffer 4NoNo1005010100KpnI-HFR314237CNEBuffer 4NoNo100250100Double Digest Recommendation(s) for BamHI-HF + KpnI-HF: Digest in NEBuffer 4 at 37C.