Traditional Cloning Quick Guide

Preparation of insert and vectors

Insert from a plasmid source

• Digest plasmid with the appropriate restriction enzymes to produce a DNA fragment that can be cloned directly into a vector. Unidirectional cloning is achieved with restriction enzymes that produce non-compatible ends.

Insert from a PCR product

• Design primers with appropriate restriction sites to clone unidirectionally into a vector
• Addition of 6 bases upstream of the restriction site is sufficient for digestion with most enzymes
• If fidelity is a concern, choose a proofreading polymerase such as Q5 High-Fidelity DNA Polymerase (NEB #M0491)
• Visit www.NEBPCRPolymerases.com for additional guidelines for PCR optimization
• Purify PCR product by running the DNA on an agarose gel and excising the band or by using a spin column (NEB #T1030, NEB #T1020)
• Digest with the appropriate restriction enzyme

Standard Restriction Enzyme Protocol

* Can be decreased by using a Time-Saver Qualified enzyme.

Time-Saver Restriction Enzyme Protocol

* Time-Saver qualified enzymes can also be incubated overnight with no star activity.

Insert from annealed oligos

• Annealed oligos can be used to introduce a fragment (e.g., promoter, polylinker, etc.)
• Anneal two complementary oligos that leave protruding 5´ or 3´ overhangs for ligation into a vector cut with the appropriate enzymes
• Non-phosphorylated oligos can be phosphorylated using T4 Polynucleotide Kinase (NEB #M0201)

Typical Annealing Reaction

Vector

• Digest vector with the appropriate restriction enzymes. Enzymes that leave non-compatible ends are ideal as they prevent vector self-ligation.

Dephosphorylation

• Dephosphorylation is sometimes necessary to prevent self ligation. NEB offers four products for dephosphorylation of DNA:
  
  • Quick CIP (NEB #M0525), Shrimp Alkaline Phosphatase (rSAP) (NEB #M0371) and Antarctic Phosphatase (AP) (NEB #M0289) are heat-inactivated phosphatases.

Dephosphorylation of 5´ ends of DNA using Quick CIP

Dephosphorylation of 5' ends of DNA Using Shrimp Alkaline Phosphatase (rSAP)

Note: Scale larger reaction volumes proportionally.

Blunting

• In some instances the ends of the insert or vector require blunting
• PCR with a proofreading polymerase will leave a predominantly blunt end
• T4 DNA Polymerase (NEB #M0203) or Klenow (NEB #M0210)  will fill in a 5´ overhang and chew back a 3´ overhang
• The Quick Blunting Kit (NEB #E1201) is optimized to blunt and phosphorylate DNA ends for cloning in less than 30 minutes
• Analyze agarose gels with longwave UV (360 nM) to minimize UV exposure that may cause DNA damage

Blunting with the Quick Blunting Kit

* PCR-generated DNA must be purified before blunting using a commercial purification kit (NEB #T1030), phenol extraction/ethanol precipitation or gel electrophoresis and subsequent extraction (NEB #T1020)

Phosphorylation

• For ligation to occur, at least one of the DNA ends (insert or vector) should contain a 5´ phosphate
• Primers are usually supplied non-phosphorylated; therefore, the PCR product will not contain a 5´ phosphate
• Digestion of DNA with a restriction enzyme will always produce a 5´ phosphate
• A DNA fragment can be phosphorylated by incubation with T4 Polynucleotide Kinase (NEB #M0201). T4 PNK can be inactivated at 65°C for 20 minutes.

Phosphorylation With T4 PNK

Purification of Vector and Insert

• Purify the vector and insert before ligation by either running the DNA on an agarose gel and excising the appropriate bands or using a spin column (NEB #T1020, NEB #T1030)
• DNA can also be purified using β-Agarase I (NEB #M0392) with low melt agarose or an appropriate spin column or resin
• Analyze agarose gels with longwave UV (360 nM) to minimize UV exposure that may cause DNA damage

Ligation of Vector and Insert

1. Use a molar ratio between 1:1 and 1:10 of vector to insert (1:3 is typical). Use NEBioCalculator to calculate molar ratios.
2. If using T4 DNA Ligase (NEB # M0202) or the Quick Ligation™ Kit (NEB #M2200), thaw and resuspend the Ligase Buffer at room temperature. If using Ligase Master Mixes, no thawing is necessary.
3. The Quick Ligation™ Kit (NEB #M2200) is optimized for ligation of both sticky and blunt ends
4. Instant sticky-end Ligase Master Mix (NEB #M0370) is optimized for instant ligation of sticky/cohesive ends
5. Blunt/TA Ligase Master Mix (NEB #M0367) is optimized for ligation of blunt or single base overhangs, which are the more challenging type of ends for T4 DNA Ligase
6. Following ligation, chill on ice and transform
7. DO NOT heat inactivate when using the Quick Ligation Buffer or Ligase Master Mixes as this will inhibit transformation
8. Electroligase (NEB #M0369) is optimized for ligation of both sticky and blunt ends and is compatible with electroporation (i.e., no cleanup step required)
9. Improved Golden Gate Assembly can be achieved by selecting high fidelity overhangs [Potapov, V., et al (2018) ACS Synth. Biol. 2018, 7, 11, 2665-2674, https://doi.org/10.1021/acssynbio.8b00333i] Try our Ligase Fidelity Tools. 

The following three tables show ligation using a molar ratio of 1:3 vector to insert for the indicated DNA size. Use NEBioCalculator to calculate molar ratios.

Ligation with the Quick Ligation™ Kit

Ligation with Instant Sticky-end Ligase Master Mix

Ligation with Blunt/TA Ligase Master Mix

Transformation

• If recombination is a concern, then use the recA^- strains NEB 5-alpha Competent E. coli (NEB #C2987), NEB-10 beta Competent E. coli (NEB #C3019) or NEB Stable Competent E. coli (NEB #C3040)
• NEB-10 beta Competent E. coli works well for constructs larger than 5 kb
• NEB Stable Competent E. coli (NEB #C3040) can be used for constructs with repetitive sequences such as lentiviral constructs
• If electroporation is required, use NEB 10-beta Electrocompetent E. coli (NEB #C3020)
• Use pre-warmed selection plates
• Perform several 10-fold serial dilutions in SOC or NEB 10-beta / Stable Outgrowth Medium for plating

Transformation with NEB 5-alpha Competent E. coli