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Tools for PCR & Cloning

Maximize your PCR success with BIOKÉ

BIOKÉ offers you a complete selection of enzymes, reagents, and instruments for every step of your cloning workflow. This website highlights a selection of key products for each stage.

  • Amplify
  • Purify
  • Clone
  • Analyze
  • Protocols

Reaction set-up - Reagents

BIOKÉ offers a wide selection of high quality PCR enzymes and reagents to meet your research needs. Choose from a variety polymerases and nucleotide solutions from New England Biolabs (NEB). To further optimize your results, a compact, automated high-precision pipetting workstation from 4titude will increase accuracy for low-volume PCR and save up to 50% in reagent costs. BIOKÉ offers all you need for successful PCR.

OneTaq® DNA Polymerase from NEB

Robust amplification across a wide range of templates

OneTaq® and OneTaq® Hot Start DNA polymerases provide an optimized blend of Taq and Deep VentR® DNA polymerases. Both are available as stand-alone enzyme or in a master mix format. >> More information

Benefits

  • Increased fidelity - 2X higher than Taq alone
  • Robust amplification
  • Convenience - Minimal optimization, regardless of a template’s GC content

 

Phusion® High-Fidelity DNA Polymerase from NEB

The well-known high-fidelity polymerase

Phusion® High-Fidelity DNA Polymerase is trusted for many years and offers robust performance for a wide range of templates. The fidelity is >50 times higher than Taq, what means very low error rates. >> More information

Benefits

  • Fidelity – High fidelity (> 50X higher than Taq)
  • Versatility – Can be used for routine PCR as well as long or difficult templates
  • Amplicon length – Robust amplifications up to 20 kb for simple templates


 

Q5® High-Fidelity DNA Polymerase from NEB

The new standard for robust performance plus high fidelity

Q5™ High-Fidelity DNA Polymerase sets a new standard for both fidelity and performance. With the highest-fidelity amplification available (>100 times higher than Taq), Q5™ DNA Polymerase results in ultra-low error rates. The one single Q5™ reaction buffer is designed to provide superior performance with minimal optimization across a broad range of amplicons, regardless of GC content. Q5™ is the most robust and quickest high-fidelity polymerase on the market.  >> More information

Benefits

  • Fidelity – Highest fidelity (>100X higher than Taq)
  • Coverage – Superior performance for a broad range of amplicons (from high AT to high GC)
  • Speed – Shortest extension times: 10 s/kb for simple templates and 10-30 s/kb for complex templates
  • Amplicon length – Robust amplifications up to 20 kb for simple templates
  • Convenience – Normal and Hot Start enzyme available as stand-alone or in master mix format


To find the perfect polymerase for your PCR reaction, check out the Polymerase Selection Chart.

Nucleotides from NEB

Quality deoxynucleotide and ribonucleotide solution sets and mixes are also available from NEB.  >> More information

Reaction set-up - Plates

BIOKÉ brings you the best plastics for your PCR reactions: two-component PCR plates, strips, and break-away plates 

FrameStar® PCR plates from 4titude

Optimum PCR results plus easy and reliable handling

FrameStar® PCR plates combine the advantages of thin-walled polypropylene tubes for optimum PCR with a rigid frame portion for more reliable handling. In contrast to standard plastics, the rigid two-component frame technology maximizes thermal stability of plates at high temperatures. The reduced thermal expansion minimizes sample loss due to evaporation from corner positions and outer rows of wells during PCR, allowing you to downscale reagent volumes to increase efficiency and to lower your reagent costs. FrameStar® PCR plates are available for a wide range of PCR cyclers, qPCR cyclers and sequencers. >> More information
 

Benefits 

  • Better results – minimize sample loss due to evaporation
  • Save Money – Downscale reagent volumes
  • Convenient – Rigid frame with thin-walled polypropylene tubes

 

FrameStar® Break-A-Way from 4titude

Tube strips or plates – the choice is yours

FrameStar® Break-A-Way is a 96-well PCR plate that can easily be divided into smaller strips of 8 tubes, ensuring no tubes are wasted. >> More information
 

Tear-A-Way™ PCR plate from 4titude

Separate the strips easily and quickly

Tear-A-Way™ plates allow for the most flexible and efficient use of a PCR plate. No need to run half-empty plates or to cut plates with scissors. Cutting plates risks perforating wells, damaging sealing rings and contamination.. >> More information

  

Reaction set-up - Pipetting



 

4LAB™ Automated Low Volume Liquid Handling from 4titude

Affordable, easy-to-use, and precision

The 4LAB™ is an automated, high-precision pipetting system specifically designed for low-volume PCR/qPCR reaction set-up. It is available as a 4-position and a 6-position workstation. For good PCR results, small volumes of reagents need to be dispensed accurately and consistently - however manual reaction setup is tedious, time consuming and prone to human error, and most robotic systems are very expensive and complex to program. The intuitive set-up and programming of the 4LAB™ is designed for researchers without prior robotic experience and will save your laboratory time and money from day 1. >> More information

Benefits

  • Money saving – Affordable and it reduces reagent costs by using low volume PCR plates
  • Easy and Convenient Use – Intuitive software comes with built-in protocols that can be quickly and easily modified.
  • Easy Service – Pipetting modules are simple to remove.
  • Flexible – Available as 4-position and a 6-position workstation.
  • Accuracy and Precision – Excellent results for qPCR standard curves and replicates.

Reaction set-up - Sealing

4s3™ semi-automatic Thermal Sealer from 4titude

Eliminate sample loss and reduce reaction volumes

The 4s3™ is a semi-automatic thermal sealer compatible with a wide range of seals and plate designs and heights. Temperature and time settings are easily optimized to produce a 100% seal, this minimizes evaporation and eliminate sample loss. Results are more reliable and uniform, and you can save on save on reagent costs as reaction volumes can be reduced to as little as 10 μl. >> More information

Benefits

  • Flexible - Compatible with all PCR plate formats
  • Save time - Always a 100% seal
  • Save Money - Eliminates sample loss and reduces your reaction volume

PCR Reaction

BIOKÉ is committed to enhancing your PCR workflow with the latest and most innovative thermocylers.

Labcycler from SensoQuest

The most innovative thermocyclers The SensoQuest team has developed and produced thermocyclers since the 1990s. The Labcycler range represents the latest generation of cyclers, with many new and innovative features. The labcycler is available in different sizes and with different thermoblocks like the Triple Block 3x21 and the thermoblocks 48, 96 and 384.  >> More information

 

Benefits

  • Highest accuracy - 6 separately controlled peltier elements with zone temperature regulation, continuously self-calibrating temperature measuring circuitry, and automated lid
  • Intuitive interface - Large TFT Touch Screen: quick and easy protocol programming
  • Flexibility - Flexible block changing system
  • Reliable - 5 years warranty

Formats

Labcycler -  the thermocycler with the flexible block changing system (more)

  • Gradient or without
  • Different Thermoblocks available (48, 96, 384 and a triple block with 3 x 21 wells) (more)

Labcycler 48 - the little brother of the Labcycler. The thermoblock is delivered with the instrument (more) 

  • Gradient or without
  • Available with aluminium or silver 48 wells block

DNA Clean-up

NucleoSpin® Gel & PCR Clean-up Kit from MACHEREY-NAGEL

The easiest way to purify DNA from gels or PCR reactions

For optimal restriction or ligation, DNA needs to be cleaned up after the PCR reaction. The two-in-one NucleoSpin® Gel & PCR Clean-up kit is the easiest way to purify DNA fragments either directly from PCR reactions or after separation on agarose gels. There is a convenient single buffer for both reactions, and since the elution volume is only 15 μl there is generally no need to concentrate the DNA further.  >> More information

Benefits

  • Convenience - One buffer for both PCR clean-up and gel extraction
  • Time Saving - Elution volume only 15ul, no extra steps needed to concentrate

 


NucleoSpin® Gel and PCR Clean-up covers all polymerase buffer systems

A PCR fragment with a size of 165 bp was amplified using different DNAPolymerase reaction mix formulations (a–c). Additional primers were added and the mixture was purified using competitor kits from Q, S, and R. The elution was performed with 25 μL. For analysis the complete eluate was loaded onto a 1% TAE agarose gel (u: unpurified). In comparison to MACHEREY-NAGEL NucleoSpin® Gel and PCR Clean-up (MN) all other kits show lower recovery or inefficient removal of primers. With MN only 1 kit is needed instead of a separate kit for the gel and a kit for the PCR.

Restriction Digestion

HF™ Restriction Enzymes from NEB

Experience a new level of flexibility in reaction conditions

More than 200 Restriction Enzymes from New England Biolabs are 100% active in the single CutSmart Buffer, which includes BSA for added simplicity. The engineered line of High Fidelity (HF™) restriction enzymes have the same specificity as their established counterpart with the benefit of reduced star activity and rapid digestions.  >> More information

Benefits

  • One-buffer Convenience – Ideal for double digests
  • High Performance – Significantly reduced star activity, even after O/N incubation
  • High Speed – Quick set-up and fast digestions (5-15 min)



EcoRI-HF™ shows significantly reduced star activity, as compared to enzyme from other manufacturer that digest DNA in 5 minutes.

EcoRI-HF™ shows no star activity when used in 5 minutes or overnight. 50 μl reactions were set up using 1 μg of Lambda DNA, 1 μl of enzyme and recommended reaction buffer. Digests were incubated at 37°C. Marker M is the 1kb DNA Ladder (NEB# N3232).



Alternative Cloning:
Gibson Assembly
Site-Directed Mutagenesis

Ligation

It may sometimes be necessary to dephosphorylate DNA to prevent self ligation, or blunt the ends of the insert or vector before ligation. NEB offers products for dephosphorylation, blunting, and a wide variety of ligases.

Shrimp Alkaline Phosphatase from NEB

Specific and 100% inactivation at 65°C

rSAP nonspecifically catalyzes the dephosphorylation of 5´and 3´ends of DNA and RNA phosphomonoesters but does not degrade diphosphate and triphosphate linkages. It is used to prepare fragments fragments for endlabeling or to modify vector DNA to prevent recircularization. rSAP is completely and irreversibly inactivated by heating at 65°C for 5 minutes, thereby making removal of rSAP prior to ligation or end-labeling unnecessary.  >> More information
 

Benefits

  • Convenient - Heat inactivation in 5 min
  • Robust - Recombinant and high activity
  • Easy - No additives needed

Quick Blunting™ Kit from NEB

Blunting in less than 30 minutes at room temperature

The Quick Blunting™ Kit is used to convert DNA with incompatible 5 or 3 overhangs to 5 phosphorylated, blunt-ended DNA for efficient blunt-end ligation into DNA cloning vectors.  >> More information

 

Benefits

  • Fast - Restriction enzyme digested DNA is blunted in less than 30 minutes
  • Convenient - Reactions are performed at room temperature in a ready-to-use mix
  • Flexible - Suitable for restriction enzyme digested DNA, sheared or nebulized DNA, or PCR-product
     

Ligases from NEB

Extensive selection of performanceoptimized DNA ligases

With over 37 years of experience in the development and production of enzymes for molecular biology, NEB offers 10 different DNA ligase products – more than any other supplier. This extensive selection of high-quality, and performanceoptimized DNA ligases and ligase master mixes will streamline your cloning experiments.  >> More information

 

Benefits

  • Save time - Fast ligations
  • Ensure successful ligations - Robust ligation efficiency
  • Maximize your transformation efficiencies - High specificity ligation
  • Experience extreme purity – High and consistent level of purity
     

Transformation

Competent Cells from NEB

Find the perfect cells for your needs

For cloning experiments choose from several high efficiency competent cell strains. For example the NEB Turbo Competent E. coli is a chemically competent E. coli cell line suitable for high efficiency transformation and rapid colony growth.  >> More information

Benefits

  • Fast - Colonies visible after 6.5 hours
  • Reliable - Free of animal products
  • Efficient - 1-3 x 109 cfu/μg pUC19 DNA

 

DNA Isolation

NucleoSpin® Plasmid EasyPure from MACHEREY-NAGEL

Ultra-fast small scale preparation of high-purity plasmid DNA

The NucleoSpin® Plasmid EasyPure kit combines customer needs for highest plasmid yield with a very fast procedure. This columns offer a binding capacity of 35 µg plasmid DNA and allow reliable yields even from higher culture volumes. >> More information

Benefits

  • Fast procedure - 6 mini preps in 14 min
  • High capacity- Up to 35 μg of plasmid DNA

 

High plasmid yield even with increasing culture volumes. Plasmid DNA (pcDNA3.1) was isolated from 1 mL, 2 mL, 3 mL, and 4 mL culture volumes (E. coli strain DH5α) using NucleoSpin® Plasmid EasyPure and competitor kits. The isolation was performed according to the standard procedure from each manufacturer.



The isolated DNA was quantified by UV/VIS spectroscopy. Indicated yields are mean values of three isolations.

NucleoBond® Xtra Midi from MACHEREY-NAGEL

High-yield, large scale DNA purification

NucleoBond® Xtra is a new generation of anion exchangers for increased yield and purity. Based on the patented NucleoBond® technology, NucleoBond® Xtra typically yields ≥ 250 μg (Midi) or ≥ 1000 μg (Maxi) of ultra-pure plasmid DNA. >> More information

Benefits

  • Save your money - This technology results in 100% more yield, use a midi prep instead of a maxi prep.
  • Save your time - Achieve 2x CsCl purified transfection-grade plasmid DNA in less than 30 minutes.

 

Plasmid DNA was isolated following each manufacturer’s protocol using the maximum culture volume with high plasmid content. Yield of plasmid DNA was determined after precipitation (NucleoBond® Xtra) and after elution from the column (competitor kits) respectively.

Preparation time and plasmid yield – comparison to silica-membrane based kits
Kit Plasmid
Yield (μg)		 Preparation
Time (min)
NucleoBond® Xtra Midi Plus 520 28
Competitor S 300

37
Competitor P 90 41

Gel Analysis

Quick-Load® 100 bp & 1 kb DNA Ladder from NEB

Pre-mixed and ready to load

Quick-Load® 100 bp and 1kb DNA Ladders are pre-mixed, readyto- load molecular weight markers containing bromophenol blue as a tracking dye. >> More information
   

Amplify


DNA Polymerase: Q5™ High-Fidelity 2x Master Mix (NEB)

Set-up
Component 50 ul reactions Final conc.
Q5™ High-Fidelity 2X Master Mix 25 μl 1X
10 μM Forward Primer 2,5 μl

0,5 μM
10 μM Reverse Primer 2,5 μl  0,5 μM
Template DNA variable  < 1.000 ng
Nuclease-free water to 50 μl

If you are using any of the Phusion® PCR Master Mix products, add 25 µl of the 2x Master Mix. Do not add dNTPs, buffer and polymerase.
 

Protocol
Cycle step Temp Time Cycles
Initial denaturation 98°C 30 s 1
Denaturation 98°C

5-10 s

 25-35
Annealing* 50-72°C 10-30 s
Extension 72°C  20-30 s/kb
Final extension 72°C 2 min

1
4-10°C hold

* Depends on primer Tm's


DNA Polymerase: Phusion® High-Fidelity DNA Polymerase (NEB)

Set-up
Component 50 ul reactions Final conc.
Phusion® DNA Polymerase 0.5 μl 1.0 units/50 μl PCR
5X Phusion® HF/GC Buffer 10 μl

1X
10 mM dNTPs 1 μl 200 μM
10 μM Forward Primer 2.5 μl 0.5 μM
10 μM Reverse Primer 2.5 μl 0.5 μM
Template DNA variable < 250 ng
DMSO (optional) (1.5 μl) 3%
Nuclease-free water to 50 μl
 

Protocol
Cycle step Temp Time Cycles
Initial denaturation 98°C 30 s 1
Denaturation 98°C

5-10 s

 25-35
Annealing* 68°C 10-30 s
Extension 72°C  15-30 s/kb
Final extension 72°C 5-10 min

1
4-10°C hold

* Depends on primer Tm's

 

Purify

The MACHEREY-NAGEL NucleoSpin Gel & PCR Clean-up kit is the two-in-one kit with optimized recovery and elution volume. With this kit you purify DNA fragments from agarose gels or directly from PCR reactions.

Clone

Restriction Enzyme: Typical Restriction Protocol (NEB)

Set-up
Component 50 ul reactions Final conc.
Restriction Enzyme 1 unit
10X Buffer 5 μl

1X 
Template DNA Variable 1 μg

Nuclease-free water

 to 50 μl

 

Protocol
Incubation 37°C for 60 minutes

Incubation

37°C for 5-15 minutes (Time-Saver. If you shorten the incubation time, please use 1 μl restriction enzyme instead of 1 unit)

 

Dephosphorylation: Antarctic Phosphatase (NEB) - optional

Set-up
Component 20 ul reactions Final conc.
Antarctic Phosphatase 1 μl 5 units
10X Buffer 2 μl

1X 
DNA Variable 1-5 μg

Nuclease-free water

 to 20 μl

 

Protocol
Incubation 37°C for 15 minutes (5’ extensions/blunt ends) or 37°C for 60 minutes (3’ extensions)

Heat Inactivate

 65°C for 5 minutes

 

Blunting: Quick Blunting™ Kit (NEB) - optional

Set-up
Component 25 ul reaction Final conc.
Purified DNA 1-19 μl up to 5 μg
10X Blunting Buffer 2.5 μl

1X
1 mM dNTP Mix 2.5 μl 0,1 mM
Blunt Enzyme Mix 1.0 μl
Sterile water to 25 μl

Protocol
1. Incubation
  1. Reactions containing restriction enzyme digested DNA are incubated at room temperature for 15 minutes. These can be blunted directly without purification.
  2. Reactions with sheared/nebulized DNA or PCR products are incubated at room temperature for 30 minutes.
  3. Blunt ligation reactions using standard T4 DNA Ligase should be incubated overnight at room temperature.
2. Immediately inactivate enzyme in the blunting reaction by heating at 70°C for 10 minutes.
3. Proceed directly to the ligation step


Ligation: T4 Ligase Buffer (NEB)

Set-up
Component 20 ul reactions Final conc.
T4 DNA Ligase 1 μl
10X T4 DNA Ligase Buffer 2 μl

 
Vector DNA (3 kb) Variable 50 ng (0.025 pmol)
Insert DNA (1 kb) Variable 50 ng (0.076 pmol)
Sterile water to 20 μl
 

Protocol
For cohesive (sticky) ends Incubate at 16°C overnight or RT for 10 min
For blunt ends or single base overhangs Incubate at 16°C overnight or RT for 2 hours

Transformation

 Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells

 

High Efficiency Transformation: Competent Cells (NEB)

Protocol
  1. Thaw cells in single-use transformation tubes on ice for 10 minutes. For 200 μl tubes, thaw on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 μl of cells into a transformation tube on ice.
  2. Add 1 pg-100 ng of plasmid DNA (1-5 μl) to cells and mix without vortexing.
  3. Place on ice for 30 minutes.
  4. Heat shock at 42°C for 30 seconds.
  5. Place on ice for 5 minutes.
  6. Add 950 μl of room temperature SOC.
  7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  8. Mix cells without vortexing and perform several 10-fold serial dilutions in SOC.
  9. Spread 50-100 μl of each dilution onto pre-warmed selection plates and incubate at 37°C or according to recommendations.

Analyze

Pick the colonies and use the MACHEREY-NAGEL NucleoSpin® Plasmid kit for rapid small scale preparation of up to 40 μg of high-purity plasmid DNA. Analyze by restriction digestion. Scale up can be performed with the MACHEREY-NAGEL kit NucleoBond® Xtra Midi in about half of the time compared to other kits based on anion-exchange chromatography.

PHUSION® is a registered trademark and property of Thermo Fisher Scientific. Phusion® DNA Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific. Q5™, Quick Blunting™ and HF™ are trademarks of New England Biolabs, Inc. OneTaq®, Deep VentR® and Quick-Load® are registered trademarsk of New England Biolabs. FrameStar® is a registered trademark of 4titude® Limited. 4s3™ and 4LAB™ are trademarks of 4titude® Limited. NucleoSpin® and NucleoBond® are registered trademarks of MACHEREY-NAGEL. DH5a™ is a trademark of Life Technologies.

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