Double Digestion with Restriction Enzymes Using Different Buffers

Double Digestion with Restriction Enzymes Using Different Buffers

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I am currently working on preparing a 9 kb sequence of DNA for restriction digestion into the pBAD30 expression vector. There are very few restriction enzymes that do not have a restriction site located on my insert, and since I am using 2 restriction enzymes in my digestion, I had little choice in choosing my restriction enzymes. The only two restriction enzymes that will work for me are Xmal and KpnI. XmaI uses CutSmart buffer while KpnI uses NEB buffer. The efficiency of XmaI in CutSmart buffer is 100% while the efficiency of KpnI in CutSmart buffer is 50%. Would it be easier to perform a double digestion using CutSmart buffer and 2x KpnI than to perform two consecutive digestions?

In my experience, a double digestion in CutSmart buffer will work perfectly well. The reaction may proceed slower, but incubate it a little longer and run a gel after the digestion - you'll see whether it has worked. The other answerers unfortunately did not mention that the best way is to check the restriction products yourself on a gel. Cut out the fragment from the gel you need (and purify) if you want to avoid undigested plasmid. See if the plasmid fragment sizes correspond to the correct cutting sites. Basic lab practice and it takes very little time!

Beware of star activity - ask a technician to sequence your fragments or plasmids to be sure it's all going well (this is good lab practice), in case you see unexpected bands on your gel that may indicate unwanted restriction activity. This is not so common, though :)

NEB double digest planner is suggesting to use 2.1 buffer for your combination of restriction enzymes (XmaI 50% and KpnI 75% activity). I would be more worried about the star activity of KpnI in CutSmart buffer than the lower activity. The latter can be overcomed by prolonging reaction time or using more enzyme.

If you haven't already ordered your enzyme, you could get high fidelity KpnI, which has been engineered for use in CutSmart.

You could also consider doing a sequential digest rather than a double digest (ie digest with one enzyme in its optimal buffer then change the buffer for digestion with the second enzyme). It is more work but benefits from easier troubleshooting in case one of your enzymes is not cutting (which can occur frequently if you have a freezer full of 10 year old enzymes).

Double Digestion with Restriction Enzymes Using Different Buffers - Biology

Restriction endonucleases such as EcoRI recognize specific palindromic sequences and cleave a phosphodiester bond on each strand at that sequence. After digestion with a restriction endonuclease the resulting DNA fragments can be separated by agarose gel electrophoresis and their size can be estimated. A restriction map is generated by using the fragment size data to determine the location of the specific endonuclease recognition sequences on the plasmid.

Each restriction enzyme requires specific reaction conditions for optimum activity. One of the most important reaction conditions which varies between different restriction enzymes is the salt (usually NaCl) concentration. Enzyme buffers are specifically formulated to provide the salt concentration for optimal enzyme activity. It is important, therefore, that the correct buffer solution is used for a particular restriction enzyme.

Listed below is a general procedure for conducting restriction digests. Keep in mind, however, that one should always consult the manufacturers recommendations of optimal conditions for each restriction enzyme (See Resources for more information on restriction enzymes).

1. For each digest combine the following solutions in a microcentrifuge tube.

Item Amount
deionized water 6 uL
10x reaction buffer 1 uL
Plasmid miniprep DNA 3 uL
Total 10 uL
2. Remove the restriction endonuclease (specific enzymes to use will be listed on the black board) from the freezer and place on ice. It is important to keep the enzyme cold. Add 0.5 uL of enzyme solution to the mixture you made in step 1. If digesting with two restriction enzymes (double digest), add 0.5 uL of the second enzyme.

3. Tap the tube to mix the contents and then pulse down the tube in a microcentrifuge.

4. Incubate at 37 deg C for 2 h to overnight. After incubation place tubes in the freezer.

The First Digest of the Semester

Your first restriction digest will be a double digest to determine the size of your insert (the cDNA). A double digest is one where two restriction enzymes are used to digest DNA in a single reaction. In this case you will be using EcoR I and BamH I. There is only one site in the plasmid vector for each of these enzymes and they are located on either side of your insert DNA.

Digesting with both will cut the insert from the vector. Next week you will determine the size of the insert by seperating the digested DNA on an agarose gel. The insert may also contain a site for one or both of these enzymes and if so, the insert will be cut into multiple pieces. By adding up the sizes of each fragment you can still determine the size of the insert.

1. In a microcentrifuge tube place 6 uL of deionized water, 1 uL of 10x EcoR I reaction buffer , 3 uL of your plasmid miniprep, 0.5 uL of EcoR I enzyme and 0.5 uL of BamH I enzyme. The enzymes and buffer are stored in the freezer. The tube with the buffer has a black top. The tube with EcoR I enzyme has an "E" with a black dot in top and the tube with the BamH I enzyme has a "B" and a brown dot on the top.

2. Tap the tube to mix the contents and then pulse down the tube in a microcentrifuge.

3. Incubate in the 37 degree incubator for 1 hour. Either store it in the freezer or prepare it for agarose gel electrophoresis.

NheI, EcoRI vector double digest - Troubling getting second enzyme to cut (Aug/21/2007 )

Hello. I'm new to cloning, and I'm trying to ligate a 1.8 kb insert into a 5.7 kb vector (modified version of pET15b). I'm trying to digest using EcoRI and NheI enzymes using different cohesive ends. If we forget about the insert, I'm having trouble digesting the vector. Both enzymes are able to cut the vector efficiently, as I can see from my gel. However, I can't seem to get the second enzyme to cut. All enzymes and buffers come from NEB. I've tried double digests as well as sequential single digests using two methods:

1. Cut the vector (from Qiagen MiniPrep from DH5A cells) using NheI in EcoRI buffer with BSA (as recommended by NEB for double digests). Incubate 37C for 2.5 hours. Heat inactivate 65C for 20 minutes. Add EcoRI and incubate again at 37C for 2.5 h. Add CIP for the last half hour, keeping an aliquot as a control without CIP. Use the Qiagen PCR cleanup kit to clean the sample, and then set up ligations. I've gotten no colonies on the transformation with CIP, but lots of colonies without CIP. I use 1 ul of enzyme in 60 ul of reaction, and then add a second ul halfway through. I use 0.5 ul CIP.

2. Cut the vector using EcoRI in EcoRI buffer. Incubate 37C for 2.5 hours as before. Use the Qiagen PCR cleanup kit. Digest with NheI, again for 2.5 h at 37C, this time in NEB buffer 2 with BSA (recommended for digests with NheI alone). Dephosphorylated with CIP as before. Cleanup with PCR cleanup kit. Volumes are the same as above. The results from the transformations are also the same.

Now, the cut sites are only separated by 6 bp (GCTAGC-ATGCAT-GAATTC). NEB says that NheI should be able to cut a vector cleaved by EcoRI with the two cut sites within 1 bp of each other on their web site, however. The way I'm interpretting the transformation results is that each enzyme will cut the vector the first time, but the second digestion isn't working. Any ideas why not?

Firstly I would simply use NEB buffer 2. There is no need to use special EcoRI buffer anymore.

Digest first with NheI, perhaps for 3hrs in either NEB buffer 2 or 4. Then add EcoRI. THere is no need to have an intermediate heat kill step or purification step. Digest for a futher 3hrs.

Find out how much DNA you have and dephosphorylate appropriately.

I believe the main cause of failure is not the digest but the dephosphorylation. Excessive dephosphorylation will damage your DNA ends and render said vector unligatable. 30 minutes is far too long (unless you have bucket loads of DNA). How much vector are you dephos?

the rule of the thumb is
1pmol DNA, is dephosphorylated by 0.1U CIP in 60mins at 37 Celsius in a volume of 50ul.

NEB CIP, is about 10U CIP per 1ul. So for most dephosphorylation I actually need make a dilution to work with.

Thanks for your response. I'll try with less CIP. But what I don't understand is if that is the problem and not the digestion, why am I getting colonies on my control plate that has the double digested vector without the CIP treatment? That's why I initially thought the problem was with the second digestion.

how many colonies are you seeing from the control plate? 100s or 10s. If the number is low, one tends to assume the restriction enzymes works. Furthermore, the enzymes that you are using are good enzymes, they behave well. So the eye of suspicion tends to fall on them last.

Typically, I'm getting in the 40-50 range. Roughly the same as the single digest ligation control (cut with one enzyme, then ligate). When I've tried to ligate the insert in, I've also gotten back the original vector that I'm trying to get the insert into.

1. It's difficult to see which one of the enzymes is not working if you only have a 6bp difference, so you need to make sure both your enzymes work - ie digest your vector with each enzyme separatly then run a gel.

2. If both enzymes work, then obviously your double digest isn't working. One possible problem could be that the glycerol concentration of the enzymes is too high in your digest, it should never be more than 10%.

3. Are you sure you have 6 bp between the cutting sites? Don't rely on what the people who gave you the vector say, unless it is a company bought plasmid. You need to have the actual sequence, so do a sequencing.

4. If you are sure that there are 6 bp and both your enzymes work separatly then you need to consider that the enzymes don't work together, irrespective of what NEB says. Then you would have to consider using other cutting sites, or maybe PCR-primer cloning or subcloning a small insert into one of the sites, so you'll have more than 6 bp in between the sites.

I tried again using a different batch of competant cells, and I'm now getting 100s of colonies on the double digestion plate without CIP dephosphorylation, and similar numbers with the EcoRI-digestion/religation control.

I've checked that both of the enzymes digest the undigested vector individually on a gel. They can both linearize the vector. I don't think I've ever exceeded 5% glycerol concentration. I do have some sequencing results that showed that there are six bp between the restriction sites, consistent with the sequence I was given. And I know that each enzyme only cuts once from the single digests as shown on the gel. NEB says that the enzymes cut within ONE bp of each other, so I'd be surprised if it couldn't manage with six, though at this point I'm not sure what else it could be.

Activity of Restriction Enzymes in PCR Buffers

Frequently, a PCR product must be further manipulated by cleavage with restriction enzymes. For convenience, restriction enzyme digestion can be performed directly in the PCR mix without any purification of the DNA. This table summarizes the percent activity of restriction enzymes on the DNA in the Taq, Phusion® or Q5® PCR mixes described below.

In these reactions, 5 units of restriction enzyme were incubated at the appropriate reaction temperature for 1 hour in a PCR mix containing 1 µg of DNA and 1 unit of DNA Polymerase in a 50 µl reaction volume with a final buffer concentration of 1X, and supplemented with dNTPs (200 µM final concentration). Enzyme activity was analyzed by gel electrophoresis.

Notes: The polymerase is still active and can alter the ends of DNA fragments after they have been cleaved, affecting subsequent ligation. Primers containing the recognition site of the restriction enzyme can act as competitive inhibitors in the cleavage reaction. The use of restriction enzymes under non-optimal conditions does increase the likelihood of star activity. If any problems are encountered, the DNA should be purified using a commercial spin column (we recommend the Monarch PCR & DNA Cleanup Kit, NEB# T1030) or by phenol/chloroform followed by alcohol precipitation.

*It has been shown that the addition of 1x restriction enzyme buffer may help to improve the ability of some enzymes to cleave.

Chart Legend: Cleavage in extension mix with 5 units of enzyme:
+++ complete cleavage ++

Enzyme Taq in std Taq Reaction Buffer Taq in ThermoPol™ Reaction Buffer Phusion in Phusion HF Buffer Q5 in Q5 Buffer* OneTaq in OneTaq Buffer LongAmp Taq in LongAmp Taq Buffer
AatII § § +++ +++ § ++ § + ++ +++ ++ +++ +++
AclI § +++ +++ § +++ +++ ++ § +++ +++ ++ § § § +++ +++ +++ + +++ § +++ +++ ++ § +++ § +++ - - - + +
AluI § +++ +++ +++ + +++ +++
AlwI § § § +++ +++ § +++ +++ § § +++ +++ +++ ++ ++ +++
ApoI-HF § +++ +++ ++ + +++ +++
AscI § +++ +++ § + +++ + § +++ +++ ++ § +++ +++ +++ § +++ +++ ++ § +++ +++ § +++ +++ +++ § - - ++ § +++ +++ +++ § +++ +++ - § +++ +++ +++ § +++ +++ +++ § +++ +++ ® § - + - - - +
BbvCI § § +++ +++ ++ § § § +++ § - - - - § +++ +++ +++ ++ +++ +++
BclI-HF § +++ +++ - - + +
BcoDI § § § § § § § § § +++ +++ ++ § ++ ++ + § § +++ +++ ++ - § § ++ +++ +++ ++ +++ +++
BsaBI @60°C § § +++ +++ +++ + +++ +++
BsaI-HF®v2 § + + + § +++ +++ ++ § § § +++ +++ ++ § +++ +++ ++ ++ +++ +++
BsgI § § +++ +++ ++ § § ++ +++ +++ ® § - - - - - -
BslI @55°C § +++ +++ +++ ++ +++ +++
BsmAI @55°C § +++ +++ +++ ++ § § § +++ +++ § +++ +++ +++ +++ ++ +++
BspCNI § § § - - § § + § + +++ ++ § § +++ +++ + § § - § § +++ +++ +++ § ++ +++ + § +++ +++ + - +++ +++
BstAPI @60°C § ++ +++ ++ § +++ +++ +++ ++ +++ +++
BstEII @60°C § +++ +++ § + +++ § +++ +++ § +++ +++ § § +++ +++ ® § +++ +++ + - +++ +++
Bsu36I § § § +++ +++ ++ + ++ ++
BtsCI @50°C § +++ +++ § - +++ + - +++ +++
Cac8I § +++ +++ § +++ ++ § § +++ +++ + § - +++ ++ § + +++ +++ + ++ +++
DdeI § +++ +++ + ++ +++ +++
DpnI § § +++ +++ +++ ++ +++ ++
DraI § ++ +++ +++ § + ++ +++ ++ ++ ++
DrdI § ++ +++ +++ § +++ +++ - § +++ + + § + +++ +++ § § ++ +++ § § +++ +++ - § § + + +++ § +++ +++ + § § + + § +++ +++ +++ - + +++
FatI @55°C § ++ ++ +++ § +++ + ++ § +++ +++ § +++ +++ + + +++ +++
FseI § § + § +++ +++ +++ § +++ +++ +++ § +++ § +++ +++ +++ § +++ +++ § ++ +++ + § + +++ § +++ +++ +++ +++ + +++
HinP1I § +++ +++ +++ + +++ +++
HpaI § +++ +++ +++ § + +++ § +++ § + +++ ++ - + ++
HpyCH4III § § ++ +++ § +++ +++ § +++ +++ + § +++ +++ + - § + +++ ++ + +++ +++
Hpy188III § § - +++ +++ § +++ +++ + ++ ++ § ++ ++ ++ - § +++ +++ +++ § +++ +++ ++ + + +
MfeI § ++ +++ § + + - - +++ § +++ + § ++ +++ ++ ++ ++ ++
MluI-HF® § ++ ++ ++ - ++ ++
MlyI § § § +++ +++ + + + +
MscI § § + § +++ +++ + § +++ +++ +++ § +++ +++ +++ § +++ +++ +++ +++ ++ +++
NaeI § § - - ++ § +++ +++ § +++ +++ + § +++ +++ - § ++ § - - + § +++ +++ - § +++ § § § +++ ++ + § ++ +++ § ++ ++ + + ++ ++
NruI-HF® § +++ ++ - - + -
NsiI § +++ +++ +++ + ++ +
NsiI-HF® § +++ +++ +++ ++ +++ +++
NspI § +++ +++ § +++ +++ § +++ +++ § + - - - +++ ++
PciI § + § +++ +++ § + + +++ § + +++ § +++ +++ + § +++ +++ § + - - - + § +++ +++ +++ § +++ +++ § + +++ - - +++ +++
PspGI @75°C § +++ +++ +++ +++ +++ +++
PspOMI § +++ +++ + § +++ +++ ++ § ++ ++ + + § ++ +++ ++ § § +++ +++ +++ § +++ +++ + § ++ + - - § +++ +++ ++ § § +++ +++ + § +++ +++ § +++ +++ +++ § § +++ + +++ § § +++ +++ § § § + + - - § + + § +++ +++ +++ +++ +++ +++
SexAI § +++ +++ +++ § - - ++ § +++ +++ § § +++ +++ +++ § § § § +++ § +++ +++ § +++ +++ § +++ +++ ++ + § +++ +++ + § + § ++ ++ + § +++ +++ § § ++ + § § +++ +++ + § § +++ +++ +++ +++ +++ +++
Tsp45I @65°C § § +++ +++ + § + + § +++ +++ ++ § +++ +++ § +++ +++ + § § +++ +++ + § +++ +++ § +++ +++
§ An HF version of this enzyme is available.

Phusion® was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific. This product is manufactured by New England Biolabs, Inc. under agreement with, and under the performance specifications of Thermo Fisher Scientific.
Phusion® is a registered trademark of Thermo Fisher Scientific.
ThermoPol&trade is a trademark of New England Biolabs, Inc.

Double Digestion with Restriction Enzymes Using Different Buffers - Biology


Background Information about Restriction Enzyme Mapping and Pencil Exercise/Questions

Plasmids are extrachromosomal, self-replicating double-stranded DNA molecules. Most plasmids exist as supercoiled molecules (CCC = covalently closed circular DNA). Although small, plasmids encode a number of important gene products. Some may confer selectable phenotypes to their recipient cells such as resistance to certain antibiotics or heavy metals, which indicate that the cell has been transfected with plasmid DNA. Other plasmid genes are essential to maintain the copy number (the number of plasmid DNA molecules per cell) or to provide origin sequences which function in the initiation of plasmid DNA replication. Generally, plasmid replication and gene expression depend entirely on the preexisting host factors required to promote these processes. Many plasmids are said to be cryptic if they do not express selectable phenotypes in cells. Such plasmids are, in general, useless as cloning vehicles since plasmid transfection is not readily assayable.

Although discovered initially in Prokaryotes, several plasmids have been isolated from, or constructed to work in, lower Eukaryotic cells such as yeast and in some plants. In addition, so-called shuttle or bifunctional plasmids have been constructed so that they can replicate in more than one species of bacteria. To be useful as a cloning vehicle for the amplification of inserted foreign genes, plasmids should be low in molecular weight, have selectable phenotypes and a number of unique restriction nuclease cutting sites.

In part I of this experiment, you will isolate two plasmids from cultures of bacterial cells. The plasmids you will be isolating are called expression plasmids. This means that they have been constructed so that, when introduced into bacterial cells under appropriate conditions, the inserted gene can be transcribed and translated by the cells and generate the protein coded by the inserted gene. The protein you will be generating is T4 lyozyme. The expression plasmid has been constructed by inserting the cDNA (complementary DNA, containing only the DNA sequences complementary to the mRNA of the gene) of T4 lysozyme downstream of a promoter in the plasmid that is inducible by a chemical called IPTG. When IPTG is present in the culture, transciption of the cDNA is initiated from the promoter and the mRNA is made and subsequently translated into T4 lysozyme protein. The two types of plasmids you will use and analyze will be discussed thoroughly with you in preparation for the laboratories.

You will do Part I of this experiment in Lab 3 . Part 1 consists of breaking open the bacterial cells and extracting and purifying the plasmid DNAs. During Lab 5 you will cleave the plasmid DNAs with restriction enzymes and separate the DNA fragments produced on agarose gels. By examining the pattern and sizes of these DNA fragments, you will be able to determine which plasmid is which.

Restriction endonuclease digestion of DNA has been extremely useful in the characterization of these molecules since the DNA can be broken down to manageable sizes using them. Because these enzymes recognize a specific nucleotide sequence in DNA, the same enzyme will always produce the same fragments of a certain DNA. Usually, the first step in the analysis of a new DNA is to construct a restriction endonuclease map using one enzyme initially, but eventually using several. In order to construct this map, it is necessary to determine the sizes of all enzyme-generated DNA fragments by agarose gel electrophoresis. A map showing the positions at which the endonuclease cuts the DNA can be created by ordering the fragments on the DNA.

The pH of the electrophoresis buffer used in running the agarose gel is 7.5, therefore, all of the DNA fragments will have a negative charge and will migrate towards the positive electrode. The smaller the size of the DNA fragment, the faster it will move through the pores of the agarose. You will find that the migration during electrophoresis is proportional to the inverse of the log of the molecular weight (i.e. migration distance = K/log MW where K is a constant for each gel condition). The DNA fragments are easily visualized during electrophoresis by including ethidium bromide (EtBr) in the gel. EtBr interchelates between double-stranded DNA and, as a consequence, fluoresces brightly when illuminated with UV light. Although we will not do this, one way to order the fragments is to digest the DNA only partially so that not all potential cleavage sites are used. The "partial" fragments are then isolated, redigested completely with the same enzyme and the resulting fragments are analyzed again by gel electrophoresis. If the "partials" are overlapping, a map will be produced. Another way to order the fragments is to completely digest the DNA separately with two different enzymes. Each limit fragment is then cleaved with the other endonuclease and the resulting fragments are analyzed again. Alternatively, you can include both enzymes in the same digestion and compare the disappearance or appearance of bands to deduce the cutting patterns (we will use this approach). As a pencil exercise, use the following data to order the fragments generated in a virtual experiment and produce a restriction map.

A solution of Virus B DNA (linear double stranded molecule) has been completely digested with EcoRI. An agarose gel of this restriction digest shows the following limit fragments: A limit fragment is what is generated when the DNA is cut at all its EcoRI sites.

Fig 1. Agarose Gel of EcoRI digest of Virus B DNA. Conveniently, these fragments have had their size determined by the use of Lambda Hind III markers-- data not shown.)

Each of these fragments was eluted from the gel and redigested with Taq I.
The following patterns were produced:

____ 7.1 Kb
____ 5.0 Kb
____ 2.9 Kb
____ 1.5 Kb ____ 2.3 Kb

Figure 2 Figure 3 Figure 4

Fig 2. Agarose gel of Taq I fragments from the 8.6 Kb EcoRI fragment of Virus B DNA

Fig 3. Agarose gel of Taq I fragments from the 7.9 Kb EcoRI fragment of Virus B DNA

Fig 4. Agarose gel of Taq I fragments from the 2.3 Kb EcoRI fragment of Virus B DNA

In addition, a Taq I digest was done on the intact viral DNA, giving rise to the following limit fragments:

Fig 5. Agarose gel of Taq I fragments of Virus B DNA

1. Using the data given above, construct a restriction map showing the locations of all Taq I and EcoRI sites and giving the distances (in Kb) between them. Remember that the bands shown represent the sizes of the DNA pieces made after cutting the DNA with a restriction endonuclease.

2. For this experiment, you will be doing a several double digests. Think about how this differs from the technique described above. Diagram a gel of the restriction fragments that would arise from a double digest using TaqI/EcoRI of the Virus B DNA. What do you perceive to be the advantages or disadvantages of each technique?

3. How do these approaches (double digest, limit digest/elution/redigestion with a different enzyme) compare to partial digests?

4. Many viral and bacterial DNA molecules are circular rather than linear. How would this influence the degree of difficulty experienced in construction of a restriction map? For purposes of illustration, change our linear Virus B DNA molecule into a circular molecule by ligating the ends. What happens to the various patterns of fragments generated? Show using gel patterns and a restriction map. (Assume ligation does not generate a Taq I or EcoRI site.)

Double Digestion with Restriction Enzymes Using Different Buffers - Biology

Examples of class II restriction enzymes

Haemophilus aegytius

Haemophilus influenzae Rd

Restriction enzymes are obtained from many prokaryotes and about 1500 enzymes with known sequence recognition sites have been isolated. Naming these endonucleases follows a system proposed by Nathans and Smith. Each name contains at least one capital letter and two small letters followed by a Roman numeral. The letters are initials of the genus and species of origin and the number represents the number of enzymes discovered in the organism. (Historically the numeral identified the protein peak in which the enzyme eluted during chromatography.) Additional information may be added as a letter. For Eco RI, the R indicates the particular strain of E. coli.

Restriction enzymes from different organisms may recognize the same DNA sequence. If the enzymes recognize the same site and cleave at the same position, they are labeled isoschizomers. Ones that recognize the same site but cleave in different positions are heteroschizomers or neoschizomers.

Isoschizomer example

Streptomyces phaeochromogenes

Bacillus sp. Bu 17091

Heteroisoschizomer or neoschizomer example

Xanthomonas malvacea rum

Iso- or heteroisoschizomers add flexibility to experimental design. Cost, methylation sensitivities, and types of "ends" are considerations as well as buffer conditions for optimal activity.

A few buffer conditions suit nearly all the restriction enzymes but no single buffer allows activity of every enzyme. Suppliers of enzymes always provide a reaction buffer (10x concentrate) that is optimum for the enzyme. Components of the 1x buffer usually are 10-100 mM Tris at pH 7.3 to 8.5, various levels of salts like KCl and NaCl (10 to 150 mM), 10 mM Mg(2+), 2 mM beta-mercaptoethanol. Sometimes 0.01% Triton- X100 (a detergent) and bovine serum albumin are included as a stabilizers. (Alternatively, swine skin gelatin can be used and offers the advantages that it is stable to autoclaving and costs about 1/15 as much as BSA.)

Since restriction enzymes can require different buffer conditions, some strategy must be used to do double digests. The preferred method is to simultaneously digest with both enzymes in a compatible buffer. This method can be used even if one enzyme is not fully active (e.g., 75% active). More of one enzyme can be added (e.g., 1 U of enzyme A + 1.33 U enzyme B) for equal cutting efficiency. There are limits to the excess enzyme due to increased glycerol in the reaction that can reduce specificity of some enzymes.

An alternative method is to digest with the "low salt" enzyme then add more buffer and the "high salt" enzyme to complete the digest. This obviously doubles the time required for digestion. In extreme cases the DNA can be precipitated after one digest and dissolved in the second digest buffer. Digests are carried out at 37 degrees C unless otherwise noted for the enzyme.

Non-specific or relaxed specificity cleavage or "star" activity can occur if non-optimal conditions are used. Conditions that encourage star activity include:

Digestion of bacteriophage lambda DNA using a restriction enzyme

Digestion of bacteriophage lambda DNA using a restriction enzyme.

2. Introduction :

Restriction enzymes have been described in Chapter 3 of this textbook. They are endonucleases which recognize and cleave the specific DNA sequences called restriction sites for example, £coRI (isolated from Escherichia coli) that recognizes and cleaves the sequence 5′-GAATTC-3′ to generate cohesive or sticky ends.

Similarly, Hind III isolated from Haemophilus influenzae recognizes and cleaves the sequences 5′-AAGCTT-3′ to generate cohesive or sticky ends.

Enzyme activity is represented as IU (International Unit). One unit of a restriction enzyme is the amount of enzyme required to completely digest one microgram of lambda DNA [in a reaction volume of 50 pi in one hour under optimal conditions of salt, pH and temperature (about 37°C for most restriction enzymes)].

3. Principle :

Phage lambda (λ) DNA is a liner double-stranded DNA molecule containing 48,502 base pairs (bp). Its DNA becomes circularized after release (inside the cell of E. coli) at a cohesive site called COS site. It contains five recognition sites for EcoRI and seven recognition sites for HindIII.

The complete digestion of lambda DNA with EcoRI results in 21, 226, 7421, 5804, 4878 and 3530 bp long six DNA fragments. Similarly, a complete digestion of lambda DNA with Hindlll results in eight DNA fragments viz., 23, 130, 9416, 6557, 4361, 2322, 2027, 564, 125 bp long fragments.

4. Requirements :

i. Lambda DNA, restriction enzyme such as ZscoRI or HindIII

ii. Assay buffer for restriction enzyme, sterilized water, Tips, Eppendorf tubes, micropipettes

iii. Agarose gel electrophoresis apparatus

5. Procedure

(i) Always keep restriction enzyme (EcoRI or HindIII), substrate (X DNA) and assay buffer in an ice bucket.

(ii) Take 2-5 pg of the lambda DNA as substrate in an eppendorf tube and dissolved in an appropriate volume of water.

(iii) Add 2 pi of about 10X assay buffer (available with the restriction enzyme) to the DNA in the eppendorf tube, followed by respective enzyme (5-12 units of EcoRI or 10-25 units of HindIII depending upon the amount of DNA used in the reaction step (ii).

(iv) Add sterilised water to make the final volume of reaction mixture to 20 pi. Centrifuge gently or mix by tapping with fingers.

(v) Incubate the reaction mixture for 1 hour at 37°C in a water bath or incubator.

(vi) As described in the plasmid isolation experiment, in the mean time prepare 1% agarose gel for loading and electrophoresis.

A single band is seen in the lane in which sample ‘B’ (A, DNA) has been loaded. While the second lane in which sample ‘A’ has been loaded shows multiple bands. This reveals the cleavage of sample ‘B’ by the respective restriction enzymes.

The exact number and size of the bands obtained depend on the restriction enzymes used for digesting the lambda DNA (sample ‘B’). As explained earlier, 6 DNA fragments are observed if £coRI is used and 8 DNA fragments are observed after using Hindi I).


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Double Digestion with Restriction Enzymes Using Different Buffers - Biology

Restriction enzyme digestion became a routine method of molecular biology 2 decades ago. Till now researches use restriction enzymes for cloning, analysis of genomic sequences and DNA methylation. Here I give a short overview on the usage of restriction enzymes and some tips from my practical experience.

Suppliers of restriction enzymes

At the moment there are many different companies that supply a wide variety of restriction enzymes. I have an experience with Roche, NEB, Fermentas and Gibco. All enzymes I tried are of sufficient quality therefore my final decision is based on unit price of the enzymes. At the moment most of enzymes we use are from MBI Fermentas. Some rare are from NEB. It is important to note, that NEB buffer system is compatible with that of Fermentas.

Digestion of DNA with a single enzyme

If you want to digest plasmid DNA with one enzyme than you usual protocol will look like this:

DNA up to 1 µg
Buffer (10x) 2 µl
Enzyme (10U/µl) 1 µl
H2O to 20 µl

To prepare this reaction you first pipet together DNA, buffer and water, vortex the mixture and add the enzyme. Mix by pipetting and incubate at least 40 min at the temperature optimal for the enzyme (remember, that most, but not all enzymes work at 37°C).

If you want to digest genomic DNA, your assay will look somewhat different. Two things are important. 1) usually you have to digest more DNA, therefore you have to set your reaction in a bigger volume (5-10 µg in 100 µl final volume) and 2) you will need more time for complete digestion (it can be even 18 h). Typical reaction I used to digest human genomic DNA for Southern blot was

DNA 10 µg
Buffer (10x) 10 µl
Enzyme (10U/µl) 5 µl
H2O to 100 µl

I incubate such reaction overnight at optimal temperature. Note, that it is better to use an incubator instead of a heating block, since in the block you will have condensation of water on the lid of your tube and this will destroy the reaction. If DNA has to be digested for bisulphite treatment I incubate the samples for a shorter time, since usually I don&rsquot need a complete digestion in this case.

Digestion of DNA with 2 or more different enzymes

In the case of double or triple digestion you have 2 possibilities

Use simultaneous digestion

If you can find a buffer in which all enzymes have sufficient activity (usually not lower than 50%), you can set your digestion will all enzymes simultaneously. It is important that the total volume of enzymes you add to your reaction is not more than 1/10 of the total reaction volume. The reason for this is that some enzymes have star activity if the concentration of glycerol in the reaction exceeds 5%.

For plasmid DNA your digestion reaction will look like this

DNA up to 1 µg
Buffer (10x) 2 µl
Enzyme 1 (10U/µl) 1 µl
Enzyme 2 (10U/µl) 1 µl
H2O to 20 µl

If you do not have a buffer in which all your enzymes function properly you will have to make a buffer exchange. This can be done in several different ways.

The most trivial way is to digest your DNA with one enzyme, then purify it with any kit for purification of DNA out of gel slice and set a reaction with the second enzyme. However in this case you have some losses of your DNA and as well you consume (probably unnecessary) purification reagents.

You can perform you double digestion sequentially using buffer adjustment. This is possible when your first enzyme (enzyme I) functions in a low salt buffer (buffer B from Fermentas, for example) and the second one (enzyme II) functions in a high salt buffer (buffer R or O from Fermentas). In this case you first set a reaction with enzyme I in a smallest possible volume.

DNA 1 µl
Buffer B 1 µl
Enzyme I 1 µl
H2O 7 µl

You perform the digestion for 1 h and then inactivate your enzyme (check in the table, usually 65°C for 10-15 min). Then you set a second step reaction as follows

Reaction 1 10 µl
Buffer R 5 µl
Enzyme II 1 µl
H2O 34 µl

Perform your digestion for 1 h and analyze the result on a gel.

Below is the table that shows you what happens in your reaction tube. As you can see, there is some increase in concentrations in final reaction, but this does not influence the activity of the enzyme.

Experimental overview

Today&rsquos procedures involve isolating plasmid DNA and digesting DNA with restriction enzymes. Make sure that you use the appropriate pipettor and set the volume correctly&mdashif you&rsquore unsure, then ask. Record all procedures and data in your lab notebook, indicating &ldquowho&rdquo performed a specific procedure step turn in copies of notebook pages at the end of the laboratory session.

SPECIAL NOTE: Record enough procedure details in your notebook during lab today so that you can repeat these procedures using your notebook as the ONLY resource. Write the methods in your own words (i.e., do not just &ldquocopy&rdquo the steps from the web pages or handouts).

ADDITIONAL DETAILS: for each centrifugation, record time, rcf (# x g), and temperature in your lab NB ALL centrifugations performed in the microcentrifuges are at &ldquoroom temperature&rdquo (indicate as such but don't need to report in degrees C)

A) Plasmid DNA mini prep

  • Disposal of Waste: discard bacterial supernatant and collect contaminated tips in a small beaker we will add bleach to 10% for 10 minutes before dumping into the trash and sink
  • Place culture tubes in the clear Biohazards bag these bags will be autoclaved prior to placement in the household trash
  • After the bacteria are lysed, tips, vials, and other materials should be discarded in the regular trash and sink

B) Restriction enzyme (RE) digests of plasmid DNA

Just for Fun

We would like to thank New England Biolabs for their generous support of our laboratory program

Watch the video: Ένζυμα - βιολογικοί καταλύτες (June 2022).


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