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Kits: Complete Protocol for Dovetail PCR Product Cloning Kit
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Dovetail™ PCR* Product Cloning Kit

#K1911
Lot: ___ Exp.: ___
Quantity:
for 10 reactions

#K1912
Lot: ___ Exp.: ___
Quantity:
for 30 reactions

IMPORTANT!
Store at -20°C.
Repeated freeze-thawing diminishes the quality.

*The Polymerase Chain reaction (PCR) process is covered by patents owned by F. Hoffmann-La Roche, Inc. See complete license declaimer.

 
DESCRIPTION

Dovetail™ PCR Product Cloning Kit provides a versatile and efficient method for cloning virtually any PCR amplified DNA fragment into a desired vector.

One of the most popular and effective methods to clone PCR products is introduction of a unique restriction endonuclease site at the 5'-end of each amplification primer followed by digestion of the PCR fragment with the restriction endonucleases to form the desired sticky ends for cloning. A major advantage of this approach lies in the fact that it is independent of whether the PCR products are blunt-ended or contain one or more extra nucleotides at their 3'-end. Therefore, PCR fragments obtained with any known thermostable DNA polymerases can be used.

However, a major limitation of this approach becomes apparent when the termini of a PCR fragment are to be tailored to suit the cloning site. The variety of cloning tasks demands that a large selection of distinct DNA cleavage specificities is available. Each restriction endonuclease, not excluding izoschizomeric enzymes, has its individual requirements for optimal reaction conditions and the minimal number of nucleotides to flank the target site at the end of a linear double-stranded DNA fragment. Since for the majority of restriction enzymes the latter parameter of is not provided by vendors, and because it is sometimes difficult to select proper reaction conditions for multiple digests, planning and performing a cloning experiment may be tricky and sometimes formidable.

Dovetail™ PCR Product Cloning Kit offers a versatile and convenient alternative that retains all the advantages of the method but is free from the aforementioned problems. The underlying idea of this novel method is the use of three well characterized type IIS restriction endonucleases, BpiI, Eco31I and Esp3I. These enzymes recognize hexanucleotide targets in DNA and cleave aside from their recognition sites producing four-base 5’-overhangs. Since the recognition and cleavage sites are separated in space, they are entirely independent of each other. Therefore, by selecting an appropriate flanking sequence in the 5’-section of a PCR primer, the sequence of the tetranucleotide overhang produced upon cleavage with one of the three Dovetail™ enzymes can be selected at will to match cloning sites of more than 47 restriction endonucleases (see the list*). In addition, the restriction endonuclease cleavage will remove its own recognition sequence from the resulting DNA such that only the desired portion of the PCR product and the four-base 5'-overhangs are present. In fact, two different 5’-overhang sequences matching cloning sites of two restriction endonucleases (e.g. HindIII and BamHI) on each end of a PCR fragment can be obtained using a single restriction endonuclease in one digestion reaction (see Figure 1). 

Illustration of PCR product cloning procedures
Fig.1. Illustration of Dovetail™ PCR product cloning procedure

Thus the PCR product can be inserted unidirectionally into any suitably prepared vector. Primers for PCR can be designed in such a way that the hexanucleotide vector cloning site is either retained or removed in the final ligation product. A selection of three restriction enzymes in the kit serves to enhance chances that at least for one of them the hexanucleotide recognition sequence will not be present within a desired DNA fragment. Since a hexanucleotide site occurs on average once every 4096 nucleotides in random sequence, a choice of three enzymes warrants that for a 1.6 kb PCR fragment a suitable enzyme will be found in 90% of cases.

Dovetail™ PCR Product Cloning Kit is efficient, reliable and simple to use. The experimental procedure has been designed such that unified reaction conditions are used with all three restriction endonucleases. Using this buffer, efficient cleavage of PCR products is achieved with primers containing a one-base 5’-terminal extension preceding the appropriate hexanucleotide endonuclease recognition site (see Figure 1 above).

AarI, Acc65I, Alw26I, Alw44I, BamHI, BclI, BcuI, BfmI, BglII, BpiI, BseDI, BseXI, BshNI, BshTI, Bsp120I, Bsp143I, Bsp1407I, BspTI, CfrI, Cfr9I, Cfr10I, Eco31I, Eco52I, Eco88I, Eco130I, EcoRI, Esp3I, HindIII, Kpn2I, LweI, MboI, MluI, MunI, NcoI, NheI, NotI, PagI, PauI, Pfl23II, PsuI, SalI, TasI, TatI, XapI, XbaI, XhoI, XmaJI.
 

COMPONENTS OF THE KIT

  1. Restriction Endonuclease BpiI, 10u1/µl:
    20µl (45µl) of enzyme in storage buffer: 10mM Tris-HCl (pH 7.5), 100mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA and 50% glycerol.
  2. Restriction Endonuclease Eco31I, 10u1/µl:
    20µl (55µl) of enzyme in storage buffer: 10mM Tris-HCl (pH 7.5), 200mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA and 50% glycerol.
  3. Restriction Endonuclease Esp3I, 10u1/µl:
    20µl (45µl) of enzyme in storage buffer: 10mM potassium phosphate (pH 7.4), 100mM KCl, 1mM EDTA, 7mM 2-mercaptoethanol, 0.2mg/ml BSA and 50% glycerol.
  4. 10X Buffer Y+/Tango™:
    200µl (600µl) of 330mM Tris-acetate (pH 7.9 at 37°C), 100mM magnesium acetate, 660mM potassium acetate, 1mg/ml BSA.
  5. 100mM DTT Solution:
    15µl (45µl) of 100mM DTT solution.
  6. Control PCR Product:
    for 2 (5) control reactions, 825 bp, 20µl (50µl), 0.1µg/µl.
  7. pUC19 DNA /HindIII+BamHI, Dephosphorylated:
    for 3 (5) control ligation reactions, 10µl (15µl), 0.05µg/µl.
  8. T4 DNA Ligase, 2u2/µl:
    15µl (35µl) of enzyme solution in storage buffer: 10mM Tris-HCl, pH 7.5 (25°C), 1mM DTT, 50mM KCl, 0.1mM EDTA and 50% glycerol.
  9. 10X Ligation Buffer:
    60µl (180µl) of 400mM Tris-HCl, 100mM MgCl2, 100mM DTT, 5mM ATP, pH 7.8 at 25°C.
  10. Deionized Water:
    1.5ml of water deionized on a Milli-Q® system.

1 One unit of enzyme is the amount required to hydrolyze 1µg of lambda DNA in 60 minutes in a total reaction volume of 50µl.
2 One unit of enzyme catalyzes the conversion of 1nmol [32PPi] into Norit®-adsorbable form in 20 minutes at 37°C (Weiss unit).
 

PROCEDURES

 I. Design of PCR Primers

 
1.1. The Choice of Restriction Endonucleases

Prior to selection of the PCR primers, it is important to choose the right restriction endonuclease. Either of the three restriction endonucleases (BpiI, Eco31I and Esp3I) can be used for digestion of a PCR products. The only limitation is that there is no internal site for the selected restriction enzyme.
In cases when the sequence of a template is not known it can be helpful to take into account the average DNA fragment size generated upon digestion of various genomes with these three enzymes (see the Appendix).

The recognition sequences for these enzymes are as follows:

BpiI (BbvII)
5'-GAAGACNN-3'
3'-CTTCTGNNNNNN-5'

Eco31I
5'-GGTCTCN-3'
3'-CCAGAGNNNNN-5'

Esp3I
5'-CGTCTCN-3'
3'-GCAGAGNNNNN-5'

 
1.2. Primer Design

The sequence of primers for PCR can be divided into 6 sections that form the recognition sequence and the digestion site for restriction endonuclease (I-V) as well as the sequence complementary to the DNA template (VI) (see Figure 2 below).

I II III IV V VI
5’- C GGTCTC N AGCT T NNNNNNNNNNNNNNNN -3’

Fig. 2. PCR primer structure (chosen restriction endonuclease is Eco31I, 4 bases overhang complementary to HindIII generated ends).

Primer design procedure step by step (see Figure 2):

I. A single extra nucleotide (G or C) of 5'-end is required for efficient cleavage of a PCR product with the restriction endonuclease.

II. Six nucleotides represent the recognition sequence of the selected restriction endonuclease.

III. Random spacer of either one (for Eco31I or Esp3I) or two (for BpiI) nucleotides.

IV. Four next nucleotides, that form 5'-overhanging ends for cloning and should match the internal tetranucleotide sequence of the enzyme used to create the vector cloning site. If PCR product is to be cloned using a single endonuclease site (e.g. HindIII), this fragment of the sequence will be the same in both PCR primers. If the cloning should be performed by using two different endonuclease sites (e.g. HindIII and BamHI, see Figure 1), then these nucleotides should be different in both PCR primers (AGCT will form the overhang complementary to the HindIII generated ends, and GATC to BamHI generated ends).

V. One-nucleotide spacer can be complementary to the template sequence, provided it is not necessary to retain the restriction endonuclease recognition sequence after PCR fragment ligation. In other case this nucleotide should be chosen to retain the recognition sequence (T in the case of HindIII).

VI. The complementary part of primer should be designed properly for good template and primer annealing. Typically it should be 16 to 25 bases long with the GC content of about 40-60%. The GC content and length should be chosen such that the melting temperatures of both primers differ by no more than 5°C. The primer sequence should not be self-complementary or complementary to the other primer in the PCR mixture. (More information about primer design is available in "Protocol for PCR").

 
1.3. Estimation of the Melting and Annealing Temperatures of Primers

Using the primers design as above we suggest PCR performing in two thermal profiles different in annealing step. Therefore two annealing (Ta) temperatures should be calculated for every primer.
Annealing temperature (Ta1, Ta2) should be approx. 5°C lower than the melting temperature. Tm1 is the melting temperature calculated for complementary part of the primer sequence (VI) and Tm2 is the melting temperature calculated for the whole sequence of chosen primer (I-VI).
The projected annealing temperatures of both primers should be similar (difference not more than 5°C). We recommend to calculate the melting temperatures using specialized computer programs where the interactions of the adjacent bases, the influence of salt concentration, etc. are evaluated.
Example for estimation of Ta of primers (used for preparation of control PCR fragment) is proposed in Figure 3.

5'-CGGTCTCAAGCTTTGGCGAAAATGAGACG-3’ primer1

Fig. 3. Calculation of primer annealing temperature (example). The underlined 3'-part of the primer is complementary to a DNA template. Computer calculated: Tm1 for the 3'-part of the primer is 47.4°C and Tm2 for the whole primer is 70.8°C. The recommended annealing temperatures are Ta1=42°C and Ta2=66°C.
 

 II. General PCR Protocol

In this chapter you can find general recommendations about preparation of reaction mixture and temperature cycling.

All solutions used should be thawed on ice, gently vortexed and briefly centrifuged.

Add in a thin walled PCR tube on ice:

Component of sample Final concentration Quantity per 100µl of reaction mixture
Sterile deionized water

variable
10X PCR buffer 1x 10µl
2mM dNTP mix 0.2mM of each 10µl
Primer 1 0.1-1µM variable
Primer 2 0.1-1µM variable
Taq DNA polymerase 2u/100µl variable
25mM MgCl2 1-4mM variable
Template DNA 10pg-1µg variable

Gently vortex the sample and briefly centrifuge to collect all drops to the bottom of the tube. Overlay the sample with 50µl of mineral oil or add an appropriate amount of wax.
Place samples in a thermocycler and start the program.
If it is necessary to optimize the PCR, we recommend to use PCR Optimization Kit (#K0162).
PCR should be performed in two thermal profiles with two calculated annealing temperatures Ta1 and Ta2 (see chapter 1.3).

Step Time Temperature Number of cycles
Denaturation
Annealing
Extension
1 minute
1 minute
1 minute
94°C
Ta1
72°C
5
Denaturation
Annealing
Extension
1 minute
1 minute
1 minute
94°C
Ta2
72°C
20-35
Final extension 7 minutes 72°C 1

An aliquot (5-10µl) of the PCR mixture should be analyzed on agarose gel. If smearing of the PCR product or inappropriate banding is observed on the gel, the PCR fragment should be extracted from the agarose gel after cleavage with restriction endonuclease.
 

 III. Cleavage of PCR Products with Restriction Endonucleases

The efficiency of digestion is known to depend greatly on the purity of PCR products. Taq DNA polymerase should be removed from reaction mixture, even traces of the enzyme can fill-in the 5’-overhangs, generated in the digestion reaction. For purification of PCR products we recommend DNA Extraction Kit (#K0513) or extraction with phenol/chloroform and ethanol precipitation.
 

3.1. Phenol/chloroform Extraction

  1. Add one volume (100µl) of phenol/chloroform/isoamyl alcohol (25:24:1) to PCR mixture. Vortex for 10sec. and centrifuge at maximum speed for 5 minutes.
     
  2. Transfer the upper aqueous phase to a fresh tube and add one volume (100µl) of chloroform/isoamyl alcohol (24:1). Vortex for 10sec. and centrifuge at maximum speed for 5 minutes.
     
  3. Repeat step 2 twice more.
     
  4. Transfer the upper aqueous phase to a fresh tube. Add 1/10 volume of 3M sodium acetate and two volumes of 95% ice-cold ethanol. Mix and cool at –20°C for 1 hour.
    Centrifuge at maximum speed for 10min.
     
  5. Pour off the supernatant and carefully wash the pellet with 100µl of 75% ice-cold ethanol. Dry the pellet and dissolve DNA in 10-20µl of water or TE buffer. Run 2µl on agarose gel and determine the approx. amount of DNA.
     

3.2. Digestion of PCR Product

  1. Prepare the following reaction mixture:
    purified PCR product (1µg) 1-25µl
    10X Y+/Tango™ buffer 3µl
    100mM DTT 0.3µl
    deionized water to 28.5µl
    selected restriction endonuclease (10u/µl) 1.5µl
  2. If primers are designed with recognition sequences for two different restriction endonucleases, it is possible to carry out double digestion in the same reaction buffer. In this case add 1.5µl of each restriction endonucleases to reaction mixture.
     
  3. Vortex the tube and spin down in a microcentrifuge for 3-5sec.
     
  4. Incubate the mixture for 1 hour at 37°C.
     
  5. Stop the reaction by heating at 65°C for 20min.

Run 5µl on agarose gel and determine the fragment size. If digested PCR fragment is shorter than PCR product more than 20 bp, it has an internal site for selected restriction endonuclease and is not suitable for ligation.
If smearing of the PCR product or inappriorate banding is observed on the gel, desired PCR fragment should be purified from agarose gel after digestion. For DNA purification from agarose gel we recommend DNA Extraction Kit (#K0513). Electroelution [1, 2], extraction with organic solvents [1], centrifugation through microcolumns [3] or digestion of agarose with agarose are also suitable.
 

3.3. Digestion of the Control PCR Product

Control PCR product, included in this kit, is obtained using primers containing Eco31I sites. Cutting this DNA with Eco31I results in a fragment with 5’-ends complementary to HindIII and BamHI generated ends.

  1. Prepare the reaction mixture in a microcentrifuge tube:
    control PCR product (1µg) 10µl
    10X Y+/Tango™ buffer 3µl
    100mM DTT 0.3µl
    deionized water 15.2µl
    restriction endonuclease Eco31I (10u/µl) 1.5µl
  2. Vortex the tube and spin down in a microcentrifuge for 3-5sec.
     
  3. Incubate the mixture for 1 hour at 37°C.
     
  4. Stop the reaction by heating at 65°C for 20min. The PCR fragment after digestion can be used in the control ligation reaction with pUC19 DNA/HindIII+BamHI, included in this kit.
     

 IV. Vector Preparation

For vector preparation restriction endonucleases leading 4 base 5’-overhangs, that are complementary to digested PCR product 5’-overhangs should be used. If PCR fragment after digestion has different 5’-overhangs, the vector should be digested with two different restriction endonucleases, resulting in 4 base 5’-overhangs, complementary to PCR fragment sticky ends. After the digestion vector should be dephosphorylated.
 

4.1. Digestion of Vector with a Single Restriction Endonuclease

  1. Prepare the following reaction mixture:
    appropriate DNA vector in water or TE buffer (0.5-4µg)   1-17µl
    10X restriction buffer 2µl
    deionized water to 20µl
  2. Add appropriate restriction endonuclease. Incubate the reaction mixture for 1 hour at recommended temperature (in general, at 37°C). Use agarose gel electrophoresis to control the completeness of the restriction reaction. In order to obtain complete restriction of the supercoiled plasmid vector, 5-10-fold restriction enzyme excess should be taken (10u/µg supercoiled DNA) or restriction time prolonged in the case, if the restriction enzyme has a tendency for the “star” activity.

If enzyme is heat sensitive, inactivate the restriction enzyme by heating at 65°C for 20min. If not, extract the DNA using DNA Extraction Kit (#K0513). Extraction with phenol/chloroform is also suitable.
 

4.2. Digestion with Two Restriction Endonucleases

Many enzymes are active in a few distinct buffers, hence it is often possible to choose a buffer that ensures sufficient activity of both enzymes ("Double Digestion of DNA with Fermentas Enzymes"). If both restriction endonucleases are active in the same buffer and at the same temperature, prepare reaction mixture with appropriate buffer and add both enzymes. Keep in mind, that the volume of restriction endonuclease added should be less than 1/10 of the volume of the final reaction mixture, because glycerol included in the enzyme storage buffer may cause “star” activity.
However, if the reaction conditions are incompatible, an easy and convenient way to perform a double digestions is to use the universal Y+/Tango™ buffer. For detailed recommendations see Double Digestion of DNA with Fermentas Enzymes using Y+/Tango Buffer.
Make sure that the cleavage reaction is complete. If the enzymes are heat sensitive, inactivate them by heating at 65°C for 20min. If they are not, extract the DNA using the DNA Extraction Kit (#K0513). Deproteinization with phenol/chloroform can also be used.
 

4.3. Dephosphorylation of the Vector 5'-ends using Calf Intestine Alkaline Phosphatase (CIAP)

  1. Dissolve the digested DNA (1-20pmol of DNA termini3) in 10-40µl of deionized water.
     
  2. Prepare the following reaction mixture:
    solution of DNA 10-40µl
    10X CIAP buffer4 5µl
    deionized water to 49µl
    CIAP5 (0.05-1u) 1µl
  3. Incubate at 37°C for 30 minutes.
     
  4. Stop reaction by heating at 85°C for 15 minutes or extract DNA with phenol and then precipitate with ethanol.
     

   Note 

The CIAP treatment can be directly performed after cleavage with a restriction endonuclease (in the same reaction mixture).

If the vector is digested only by one restriction endonuclease, after dephosphorylation it could be used for ligation directly. If it is digested with two restriction endonucleases, vector should be gel-purified to avoid religation with the small vector fragment. Gel-purification is not important if the digestion sites of restriction endonucleases are next to each other.
 

3 For estimation of ends concentrations see Appendix.
4 Any of the Fermentas Five Buffer Plus System buffers can also be used with good results.
5 Dephosphorylation of 1pmol of DNA termini requires 0.05u of calf intestine alkaline phosphatase. you can dilute CIAP with 1X buffer.
 

 V. Ligation

A factor affecting the efficiency of ligation and subsequent clone selection is the vector/insert ratio in the ligation reaction. The optimal molar ratio of the ends appears to be about 1:3, respectively [1]. We recommend using 0.18pmol ends of vector in 30µl of ligation reaction. The amount of DNA insert required for efficient ligation with 0.18pmol ends of the vector can be approximately determined from Table 1.

Table 1: Conversion table for the amount of PCR fragment DNA required for the ligation reaction.

Length of DNA fragment 
(bp)
pmoles of ends per 1µg of DNA Amount of PCR fragments for 
ligation reaction in µg (0.54pmol ends)
100 30 0.018
300 10 0.054
500 6.0 0.09
1000 3.0 0.18
2000 1.5 0.36
3000 1.0 0.54
  1. Determine the approximate DNA concentration by agarose gel electrophoresis. According to the Table 1. determine the amount of PCR fragment DNA equivalent to 0.54pmoles of ends.
     
  2. Add the following components into microcentrifuge tube:
    vector DNA (0.18pmol ends) 1-25µl
    digested PCR fragment (0.54pmol ends) 1-25µl
    10X ligation buffer 3µl
    deionized water to 29µl
    T4 DNA ligase, 2u/µl 1µl
  3. Ligate at 22°C for 1 hour. If the ligation reaction is not complete or if it is necessary to maximize the yield of useful recombinants, prolong reaction time (overnight).
     

Control ligation reaction:

In order to check the quality of the kit, the control ligation reaction may be performed with digested Control PCR product and control p