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DNA Cloning Kit

 
DESCRIPTION

The kit is designed for routine cloning of DNA fragments into DNA vectors. It provides components essential for standard cloning procedures, i.e. enzymes, reaction buffers and other solutions for ligation, filling-in of DNA recessed ends, and dephosphorylation reactions.

Successful insert/vector DNA ligation can be obtained only in the case if after the digestion compatible cohesive fragment and vector DNA ends are produced (see Fermentas Catalog 2002/03). If insert and vector sticky ends do not coincide, blunt ends suitable for ligation have to be created by filling-in 5'-protruding DNA ends with Klenow fragment, or by removing of 3'-overhangs with T4 DNA polymerase. In the presence of dNTPs Klenow fragment exhibits 5’=>3’ polymerase activity and is capable to synthesize DNA chain up to the end of the template [1]. Blunted DNA fragments are then ligated with the vector precleaved with the blunt ends producing restriction endonuclease. Recyclization of the vector molecule can be prevented by dephosphorylating 5'-ends. Most commonly, Calf Intestine Alkaline Phosphatase [1] or Shrimp Alkaline Phosphatase, are used for these reactions . Ligation of dephosphorylated vector with the DNA fragment results in the circular double-stranded DNA molecule with two nicks. This has no influence upon the transformation efficiency, since the reparation system of the cell is able of restoring intact structure of the plasmid DNA. Recombinant plasmids obtained after the ligation reaction are then introduced into competent E.coli cells.

No specific cloning vector is provided with this kit, allowing the user a free choice of any plasmid vector (Fermentas offers several cloning vectors).
 

COMPONENTS OF THE KIT

1. Klenow Fragment, 3u*/µl:
   15µl (30µl) of the enzyme solution in storage buffer: 50mM potassium phosphate (pH 7.4), 1mM DTT and 50% glycerol.
2. Buffer K (10X Klenow Fragment Buffer):
   50µl (150µl) of 0.5M Tris-HCl buffer (pH 8.0 at 25°C) containing 50mM MgCl₂ and 10mM DTT.
3. 0.25mM dNTP Mix:
   100µl (300µl) of 0.25mM dGTP, 0.25mM dATP, 0.25mM dTTP, 0.25mM dCTP aqueous solution.
4. Calf Intestine Alkaline Phosphatase (CIAP), 1u**/µl:
   15µl (30µl) of the enzyme solution in storage buffer: 20mM Tris-HCl (pH 8.0), 1mM MgCl₂, 50mM KCl, 0.1mM ZnCl₂ and 50% glycerol.
5. Buffer P (10X CIAP Buffer):
   50µl (150µl) of 0.1M Tris-HCl (pH 7.5 at 37°C) containing 0.1M MgCl₂.
6. T4 DNA Ligase, 4u***/µl:
   15µl (30µl) of the enzyme solution in storage buffer: 20mM Tris-HCl (pH 7.5), 1mM DTT, 50mM KCl, 0.1mM EDTA and 50% glycerol.
7. Buffer L (10X Ligation Buffer):
   20µl (60µl) of 400mM Tris-HCl, 100mM MgCl₂, 100mM DTT and 5mM ATP (pH 7.8 at 25°C).
8. PEG 4000 Solution:
   40µl (120µl) 50% (w/v) PEG 4000 aqueous solution.
9. Control DNA:
   60µl (6µg) (150µl - 15µg) of pUC19 DNA/HindIII fragments, 0.1µg/ml.
10. Deionized Water:
    1.5ml of water deionized on a Milli-Q® system.

* One unit of Klenow fragment catalyzes the incorporation of 10 nanomoles of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30min at 37°C using poly(dA-dT)·poly(dA-dT) as a template·primer.
** One unit of calf intestine alkaline phosphatase hydrolyzes 1 micromole of 4-nitrophenylphosphate in 1min at 37°C.
*** One unit of enzyme catalyzes the conversion of 1 nanomole of [³²PP_i] into Norit®-adsorbable form in 20min at 37°C (Weiss unit) [2].
 

TE buffer: 10mM Tris-HCl, pH 7.4; 1mM EDTA, pH 8.0.
 
TE-saturated phenol/chloroform: mix equal parts of TE buffer and phenol and allows the phases to separate. Then mix 1 part of the lower, phenol phase with 1 part of chloroform: isoamyl alcohol (24:1).
 
TAE buffer: 40mM Tris-acetate, 2mM EDTA, pH 8.0.
 
LB medium (per litre): 1% Bacto tryptone®, 0.5% Bacto-yeast extract®, 0.5% NaCl. Adjust pH to 7.5 with NaOH and autoclave. After the autoclaved solution has cooled to 50°C, add antibiotic.
 
LB plates with antibiotic: add 15g agar to 1 litre of LB medium. Autoclave. Allow the medium to cool to 55°C before adding antibiotic. Pour 30-35ml of medium into 100mm Petri dishes.
 
X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) stock solution: 20mg/ml in N,N-dimethylformamide.
 
IPTG (isopropyl-beta-D-thiogalactopyranoside) stock solution (0.1M): 1.2g IPTG, add water to 50ml final volume. Filter-sterilize and store at 4°C.
 
NaCl solution for competent cells preparation: 0.1M NaCl, 5mM Tris-HCl, 5mM MgCl₂, pH 7.0. Autoclave.
 
CaCl2 solution for competent cells preparation: 0.1M CaCl₂, 5mM Tris-HCl, 5mM MgCl₂, pH 7.0. Autoclave.
 
SOC medium: 2% Bacto tryptone®, 0.5% Bacto-yeast extract®, 10mM NaCl, 2.5mM KCl, 10mM MgCl₂, 10mM MgSO₄, 20mM glucose. Autoclave.
 

STANDARD CLONING

1. Cleavage of the DNA Vector.
2. Dephosphorylation of DNA Vector Cleaved at Unique Site.
3. DNA Fragment Preparation.
4. Filling-in of 5'-overhangs of the DNA Fragment and/or Vector Termini.
5. Ligation.
6. Transformation.
7. Clone Selection.
    

In order to obtain complete digestion of the supercoiled vector, 5-10-fold restriction endonuclease excess should be taken (10u/µg supercoiled DNA) or digestion time prolonged in the case, if the restriction endonuclease has a tendency for the “star” activity. Use agarose gel electrophoresis to control the completeness of the digestion reaction.
 

1. Dissolve digested DNA (1-20pmol of DNA termini) in 10-40µl of deionized water.
    
2. Prepare the following reaction mixture:

   solution of DNA	10-40µl
   buffer P (10x CIAP buffer)****	5µl
   deionized water up to	49µl
   CIAP ***** (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 

1. The DNA treatment with CIAP can be directly performed after the cleavage by a restriction endonuclease (in the same reaction mixture).
2. Estimation of ends (3’ or 5’) concentration:
   Circular DNA
   pmol ends = pmol DNA x number of cuts x 2
   Linear DNA
   pmol ends = pmol DNA x (number of cuts x 2 + 2)
   1µg of 1000 bp DNA = 3.04pmol ends
   1µg of linear pUC18/19 DNA = 1.14pmol ends
   1µg of linear pBR322 DNA = 0.7pmol ends
   1µg of linear SV40 DNA = 0.58pmol ends
   1µg of linear PhiX174 DNA = 0.56pmol ends
   1µg of linear M13mp18/19 DNA = 0.42pmol ends
    

**** You can successfully use any other Five Buffer Plus System buffers.
***** Usually for dephosphorylation of 1pmol of DNA termini 0.05 units of calf intestine alkaline phosphatase is used. You can dilute CIAP with 1X buffer.
 

DNA fragments suitable for ligation can be purified either electrophoretically [1] or by ultrafiltration [3] or chromatography [3, 4]. Purity of DNA fragment is important for the ligation efficiency.
Purification of DNA fragments from agarose gels is a standard procedure performed by various methods [5], such as electroelution [1, 6], extraction by organic solvents [1], binding on glass fibers [7], centrifuging in microcolumns [8] or freezing-squeezing [9]. For purification of DNA fragments we recommend DNA Extraction Kit (#K0513).
 

1. Add the following components into a microcentrifuge tube:

   DNA solution (1µg)	1-34µl
   buffer K (10X Klenow fragment buffer)	5µl
   0.25mM dNTP mix	10µl
   deionized water up to	50µl
   Klenow fragment (3u)	1µl

2. Incubate at 37°C for 10min.
    
3. Stop the reaction heating at 75°C for 10min.
    

1. Add the following components into a microcentrifuge tube:

   DNA vector solution (50ng- 0.4µg)	1-12µl
   DNA fragment solution (0.1-1µg)	1-12µl
   buffer L (10X ligation buffer)	2µl
   PEG 4000 solution (for blunt ends only)	2µl
   deionized water up to	20µl
   T4 DNA ligase (4u)	1µl (for blunt ends),
   	0.5µl (for sticky ends)

2. Incubate at 22°C for 1 hour.
    

 Note 

1. Total DNA concentration in the ligation mix should be 10-70µg/ml.
2. Mostly vector/DNA fragment molar ratio should be 1:3.
   1µg of 1000 bp DNA = 3.04pmol ends.
   1µg of linear pUC18/19 DNA = 1.14pmol ends.
3. In the ligation reactions with DNA fragments purified by gel electrophoresis, BSA up to the final concentration of 500µg/ml may be used, if necessary [1].
4. It is necessary to pre-phosphorylate the 5'-end of synthetic oligonucleotides prior their ligation with the vector. 
5. If the ligation reaction is incomplete or if it is essential to obtain the maximum yield of useful recombinants, prolong the reaction time (overnight).
6. PEG should be added when concentration of DNA vector or of insert is low.
    

The choice of strain for the competent cell preparation depends on the specific task of the experiment. Routinely, following strains are used for transformation: XL1-Blue, DH5alfa®, HB101, JM83, etc. (see Appendix in Fermentas Catalogue 2002/03 - Bacterial Strain Genotypes).
There are many contemporary ways for transformation of cells [10, 11] including traditional ones, when competent cells are prepared by metal salt treatment, such as MgCl₂ [12], RbCl and CaCl₂ [1].
For bacterial transformation we recommend TransformAid™ Bacterial Transformation Kit (#K2710,#K2711).
 

6.1. Rapid preparation of competent cells using CaCl₂

1. Transfer a single cell colony of an appropriate strain from the Petri plate into 5ml LB medium and incubate shaking overnight at 37°C.
    
2. Use 4ml of this culture to inoculate 400ml of LB medium and incubate shaking at 37°C until the culture reaches 0.3 at OD₆₀₀ for rec+ strains or 0.6 for rec- strains (about 1-3 hours).
    
3. Place culture on ice for 10 minutes and then centrifuge at 3000g for 10-15min at 4°C.
    
4. Pour supernatant off and resuspend cells in 1/2 of initial volume (200ml/400ml of culture) of sterile ice-cold NaCl solution.
    
5. Centrifuge at 3000g for 10min at 4°C.
    
6. Remove supernatant and resuspend cells in 1/2 of the initial volume (200ml/400ml of culture) of sterile ice-cold CaCl₂ solution. Store on ice for 20min.
    
7. Centrifuge at 3000g for 10min at 4°C.
    
8. Remove supernatant and resuspend cells in ice-cold CaCl₂ in 1/20 of initial volume (20ml/400ml of culture). Allow suspension to age on ice for at least 1 hour before use. Cells reach peak efficiency after 24 hours and remain competent for up to a week.
    
9. To prepare frozen competent cells, add an aqueous solution of 80% glycerol to the competent cells to the final concentration of 15-20%. Place microcentrifuge tubes on dry ice and add to each tube 200µl of competent cells. Store frozen competent cells at -70°C and thaw on ice immediately before use.
   Transformation efficiency of cells prepared this way usually ranges 10⁵-10⁶ transformants/1µg of supercoiled DNA.
    

6.2. Transformation using CaCl₂

1. Pipette sample of transforming DNA (1-20ng in 1-15µl) into suspension of competent cells (200µl) and swirl gently to mix.
    
2. Leave on ice for 30min.
    
3. Heat-shock cells at 42°C for 2min. Place on ice for 1min.
    
4. Add about 1ml of sterile LB medium to cell suspension and incubate cells at 37°C for 45min gently shaking.
    
5. Centrifuge 5000g for 2min. Take off supernatant, then resuspend the pellet and seed one to an agar plate with antibiotic, X-Gal and IPTG.
    
6. Incubate the plate overnight at 37°C.
    

6.3. Preparation of competent cells for electroporation

1. Inoculate a single colony of E.coli cells into 5ml LB medium. Grow 5 hours to overnight at 37°C with moderate shaking.
    
2. Inoculate 2.5ml of the culture into 500ml LB medium in a sterile 2-liter flask. Grow at 37°C with shaking (300rpm) to an OD₆₀₀ of 0.5-0.6.
    
3. Chill cells in an ice-water bath 10 to 15min and transfer to a 1-liter prechilled centrifuge bottle. Cells should be kept at 2°C for all subsequent steps.
    
4. Centrifuge cells 20min at 4000g, 2°C.
    
5. Pour off supernatant and resuspend the pellet in 500ml ice-cold water. Centrifuge cells as in step 4.
    
6. Pour off supernatant, resuspend the pellet in 250ml ice-cold water. Centrifuge cells as in step 4.
    
7. Pour off supernatant. If fresh cells are to be used for electroporation, resuspend the pellet in 125ml ice-cold water.
   If frozen cells are to be used for electroporation, resuspend the pellet in 125ml ice-cold 10% (v/v) glycerol. Centrifuge cells as in step 4.
    
8. Pour off supernatant. If fresh cells are to be used for electroporation, resuspend the pellet in 1.5-2ml ice-cold water.
   If frozen cells are to be used for electroporation, resuspend the pellet in 1.5-2ml ice-cold 10% glycerol. Aliquot 40 to 200µl cells into prechilled microcentrifuge tubes and freeze on dry ice. Store at -70°C.
    

6.4. Transformation of the competent cells by electroporation

1. Add equal volume of chloroform to ligation reaction mixture.
    
2. Vortex vigorously for 10sec. and microcentrifuge 30sec. at room temperature.
    
3. Carefully remove the top (aqueous) phase containing DNA and transfer to a new tube.
    
4. Repeat steps 1 to 3 twice.
    
 Important 

It is known, that the presence of active T4 DNA ligase during electroporation for some E.coli strains would interfere with the transformation efficiency of ligated DNA [20]. Therefore, the removal of the enzyme from ligated DNA by three chloroform extractions prior electroporation may be crucial for obtaining maximal transformation efficiency. The additional DNA precipitation with ethanol is recommended for DNA concentration and the maximal numbers of transformants obtaining.
 

5. In a cold 1.5ml tube mix 40-200µl of the cell suspension with 1 to 2µl of obtained DNA solution. Mix well and leave on ice for 0.5-1min.
    
6. Set the electroporation apparatus to 2.5kV when using the 0.2cm cuvettes or 1.80kV for 0.1cm cuvettes.
   Transfer the DNA and cells into a cuvette that has been chilled 5min on ice, shake slightly to settle the drops to the bottom, and wipe the ice and water from the cuvette with a filter paper. Place the cuvette into the sample chamber.
   Apply the pulse by pushing the button or flipping the switch.
    
7. Remove the cuvette. Immediately add 1ml SOC medium and transfer to a sterile culture tube with Pasteur pipette. Incubate 30 to 60min with moderate shaking at 37°C.
    
8. Plate an appropriate volume of the suspension on selective agar plates containing antibiotic, X-Gal (40µl of 20mg/ml stock solution per plate) and IPTG (40µl of 0.1M stock solution per plate).
    
9. Incubate the plates overnight at 37°C.
    

Recombinant clones are selected by white/blue selection, if the plasmid vector is lacZ genetically marked.
The presence of insert is revealed after the plasmid purification. One of the ways for plasmid isolation is alkaline lysis method [1, 13], which is applied for plasmid DNA minipreparation [14-18]. DNA purification can be performed by various methods, such as centrifugation in CsCl gradient [19], gel filtration chromatography [21-23], binding on glass powder [24] or cetyltrimethylammonium bromide (CTAB) precipitation [25]. We suggest an easy-to-use small-scale alkaline lysis modification.

1. Place 30µl TE buffer, pH 7.4 (10mM Tris-HCl, pH 7.4; 1mM EDTA, pH 8.0) into the microcentrifuge tube.
    
2. Pick up 1 cell colony with a loop, add to the tube and resuspend it.
    
3. Add 60µl of SDS-NaOH solution (1% SDS, 0.2M NaOH).
    
4. Incubate for 5min at room temperature, shaking gently occasionally.
    
5. Add 45µl of sodium acetate, pH 5.2 (3M Na-Ac, pH 5.2) and 130µl chloroform. Mix all and centrifuge in microcentrifuge for 5 minutes.
    
6. Transfer upper phase into a new tube. Add 130µl of isopropanol, mix again and centrifuge for 10min.
    
7. Remove supernatant, dry precipitate and dissolve DNA in 10-20µl TE buffer.
    

QUALITY CONTROL

1. Add the following components into the microcentrifuge tube:

   control DNA pUC19 DNA/HindIII fragments (50ng)	0.5µl
   buffer L (10X ligation buffer)	2µl
   PEG 4000 solution (for blunt ends only)	2µl
   deionized water up to	20µl
   T4 DNA ligase (4u)	(for blunt ends) 1µl,
   	(for sticky ends) 0.5µl

2. Incubate at 22°C for 1 hour.
    
3. Transform competent cells (use 4µl (10ng) of ligation reaction mixture for transformation). it is recommended to transform supercoiled pUC19 DNA for control.
    
4. Plate transformants on the plates with ampicillin, X-Gal and IPTG.
    
5. Incubate overnight at 37°C.
    
6. Count the blue colonies of transformants.
    

1. Add the following components into the microcentrifuge tube:

   control DNA pUC19 DNA/HindIII fragments (1µg)	10µl
   buffer K (10X Klenow fragment buffer)	5µl
   0.25mM dNTP mix	10µl
   deionized water up to	50µl
   Klenow fragment (3u)	1µl

2. Incubate at 37°C for 10min.
    
3. Stop reaction by heating at 75°C for 10min.
    
4. Use 2.5µl (50ng) of the resultant reaction mixture in the ligation step. Ligate as above and then transform competent cells. Use 4µl (10ng) of ligation reaction mixture for transformation. Transform cells with the supercoiled form of pUC19 DNA for the transformation efficiency control and with the pUC19 DNA/HindIII ligate (without the end filling-in).
    
5. Count white colonies..
    

1. Add into 1.5ml microcentrifuge tube:

   control DNA pUC19 DNA/HindIII fragments (1µg)	10µl
   buffer P (10X CIAP buffer)	5µl
   deionized water up to	50µl
   CIAP (1u)	1µl

2. Incubate at 37°C for 30min.
    
3. Stop reaction by heating at 85°C for 15min or extract DNA with phenol and then precipitate with ethanol.
    
4. Use 2.5µl (50ng) of the resultant reaction mixture in the ligation step. Ligate as above and then transform competent cells. Use 4µl (10ng) of ligation reaction mixture for transformation. Transform cells with supercoiled pUC19 DNA for a control.
    

Ligation efficiency: 10⁵ transformant colonies per 1µg of ligated DNA are obtained.
 

Blunting efficiency: more than 90% colonies are white.
 

Dephosphorylation efficiency: less than 10² colonies per 1µg DNA are obtained.
 

References

1. Sambrook J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989.
2. Weiss, B. et al., J. Biol. Chem. 243, 4543-4555, 1968.
3. Krowczynska, A.M et al., BioTechniques, Vol. 13, No 2, 286-289, 1992.
4. Katz, E. et al., BioTechniques, Vol. 8, No 5, 546-555, 1990.
5. Vogelstein, B. and Gillespie, D., Preparative and Analytical Purification of DNA from Agarose. Proc. Natl. Sci. USA, No 76, 615, 1979.
6. Poloquin, J.J. et al., BioTechniques, Vol. 10, No 2, 159-160, 1991.
7. Errington, J., Nucl. Acids Res., Vol. 18, No 17, 3254, 1990.
8. Schwarz, H. et al., BioTechniques, Vol. 13, No 2, 205-206, 1992.
9. Thuring, R.W. et al., Anal. Biochem. No 66, 213-220, 1975.
10. Hanahan, D., Studies on Transformation of E.coli with Plasmid., J. Mol. Biol., Vol. 166, 557-580, 1983.
11. Hanahan, D., Techniques for Transformation of E.coli. DNA Cloning. Vol1/Ed. D.M. Glover - Oxford, Washington DC:IRL Press, 109-136, 1985.
12. Alexander, L. et al., Gene, Vol. 31, No 1-3, 79-89, 1984.
13. Birnboim, H.C. and Doly, J., Nucl. Acids Res., No 7, 1513-1523, 1979.
14. Del Sal, G., Manfioletti, G. and Schneider, C., Nucl. Acids Res., No 16, 9878, 1988.
15. Morelle, G., Focus, No 11, 7-8, 1989.
16. Sounders, S. E. and Burke, J.F., Nucl. Acids Res., No 18, 4948, 1990.
17. Wang, L.M. et al., BioTechniques, No 6, 839-843, 1988.
18. (Kamal) Chowdbury, K., Nucl. Acids Res., Vol. 19, No 10, 2792, 1991.
19. Clewell, D.B. and Helinski, D.R., Proc. Natl. Acad. Sci. USA, No 62, 1150-1151, 1969.
20. Michelsen, B.K. , Analytical Biochemistry, 225, 172-174, 1995.
21. Attal, J.C. Puissant, L. and  Hondebine, L.-M., BioTechniques, No 8, 269-271, 1990.
22. Griffith, D.M., BioTechniques, No 6, 725-727, 1988.
23. Raymond, G.J., Bryant III, K., Nelson, A. and Johnson, J.D., Anal. Biochem. No 173, 125-133, 1988.
24. Marko, M.A., Chipperfield, R. and Birnboim, H.C., Anal. Biochem., No 121, 382-387, 1982.
25. Ishaq, M., Wolf, B. and Ritter, C., BioTechniques, No 9, 19-24, 1990.

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Updated August 23, 2002 15:19