Troubleshooting for Molecular Cloning

Overview of Molecular Cloning

Molecular cloning is used to assemble recombinant DNA molecules and to direct their replication within host organisms, making multiple DNA molecules. Cloning involves the replication of one molecule to produce a population of cells with identical DNA molecules. It is commonly used to amplify DNA fragments containing whole genes or any DNA sequence (such as promoters, non-coding sequences and randomly fragmented DNA). Molecular cloning is the process of inserting the gene-of-interest (GOI) into a plasmid vector and this vector is then inserted into a cell that expresses the protein encoded by the GOI. Once protein is expressed in the cell, the protein expression can be used for different studies (such as cell signaling, morphology or other aspects).

Cloning is not only creating copies of a gene or other cellular component but also changing a gene within a sequence of DNA and then replicating it. To amplify any DNA sequence in a living organism, the sequence must be linked to an origin of replication and capable of directing the propagation of itself and any linked sequence (for example: Rapid DNA Dephos & Ligation Kit can be used for cloning).

Importance/Application of Molecular Cloning

Molecular Cloning is used for the following:

• It is used to explore proteins and their function. Using the recombinant DNA, research scientists can direct the replication (production of recombinant proteins) and manipulate cells with cloning, thereby learning more about specific proteins and apply this knowledge to larger-scale research endeavors like diseases and pathogens.¹
• It helps in exploring the genomic sequence of many different species (including humans and other animals) and creating transgenic organisms like herbicide resistant plants etc.
• It is used in cell cycle, genome organization, gene expression and gene therapy. It is also useful in discovering new medicines and for the synthesis of new recombinant vaccines.
• It acts an important tool in clinical microbiology due to its simplicity, cost effectiveness, rapidity and reliability. Industrial application of this technology leads to the production of new antibiotics in the form of antimicrobial peptides and recombinant cytokines that can be used as therapeutic agents.¹
• Using this technology gene probes are developed that have many useful roles in early diagnosis of hereditary diseases, forensic investigations and routine diagnosis.¹

Process of Molecular Cloning

Cloning of any DNA fragment essentially involves the following steps:

• Choice of a cloning vector (for example: PSF-OXB20-FLUC - BACTERIAL LUCIFERASE PLASMID or PSF-OXB20-BETAGAL - BACTERIAL BETA GALACTOSIDASE PLASMID or PSF-OXB20 - STRONG BACTERIAL PROMOTER PLASMID and host organism. For help selecting a vector, see our Expression Vector Selection Guide
  
• Preparation of vector DNA (for example: GenElute™ Plasmid Midiprep Kit, Plasmid DNA from E. coli RRI)
• Preparation of DNA fragment that needs to be cloned
• Recombinant DNA creation using DNA ligase (for example: T4 DNA Ligase)
• Introduction or insertion of the recombinant DNA into the host organism:
• Insertion of DNA into the host organism depends upon the experimental method chosen (i.e. transformation, transduction, transfection, electroporation)
• Selection of organisms containing vector sequences
• Screening for clones with desired DNA inserts and with biological properties

The detailed article on molecular cloning can be found in the Molecular Biology handbook.

In the process of molecular cloning, various problems are faced and that can be checked. Here below are few possible causes and recommended solutions:

Vector Choice

Consider these four DNA segments during cloning; absence of any of these DNA segments may cause problems:

• An origin of DNA replication is essential (for replication inside host)
• One or more unique restriction endonuclease recognition sites (where foreign DNA may be introduced)
• A selectable genetic marker gene used to enable the survival of cells that have taken up the vector sequences
• An additional gene (used for screening of cells containing foreign DNA)

DNA Fragment Preparation

Incomplete Restriction Enzyme (RE) digestion

• Restriction enzyme(s) not cleaving completely: Need to check the methylation sensitivity of the enzyme(s), use the recommended buffer supplied with the restriction enzyme. Cleaning up the DNA to remove contaminants that may inhibit the enzyme. (May use the following products for better results)
  
• Salt inhibition: A few enzymes are salt sensitive, so DNA needs to be cleaned prior to digestion.
• PCR components inhibition: Need to clean the PCR fragment prior to restriction digest.
• Using of wrong buffer: Need to use only the recommended buffer supplied along with the restriction enzyme. (Double digestion: Digesting a DNA substrate with two restriction enzymes simultaneously is called double digestion and is a common timesaving procedure) For double digestion, need to choose the right buffer that result in the most activity for both enzymes. The recommended protocol should be followed. In case two different incubation temperatures are necessary, the optimal reaction buffer is supposed to be chosen and the reaction needs to be set up accordingly.
• If too few units of enzyme used: Use of least 3–5 units of enzyme per μg of DNA is generally recommended.
• Short period of incubation: Need to increase the incubation time.

The digested DNA ran as a smear on an agarose gel.

• The restriction enzyme(s) may be bound to the DNA substrate: Need to add SDS 0.1–0.5% (for example: Sodium dodecyl sulfate solution) to the loading buffer to dissociate the enzyme from the DNA.
• Nuclease contamination: Use of fresh, clean running buffer and a fresh agarose gel (for example: Agarose, low gelling temperature) may help, along with cleaning up the DNA.

Extra bands or a smear on the gel

If larger bands are seen in the gel than expected, this may indicate binding of the enzyme(s) to the substrate: May need to lower the number of units in the reaction and addition of SDS 0.1–0.5% (for example: Sodium dodecyl sulfate solution) to the loading buffer to dissociate the enzyme from the substrate may help.

Annealing temperature may be too low: Need to determine the correct annealing temperature of the primers. (For example: KiCqStart^® SYBR^® Green Primer, Hexanucleotide Primers can be used).

No amplification of PCR fragment

This may be caused due to the following reasons, which need to be checked during the experiment²:

• Use of wrong primer sequence or error in PCR primers
• Incorrect annealing and extension temperature
• Incorrect primer concentration and too few units of polymerase: Need to use the manufacturer’s recommended primer concentration and polymerase units based on the reaction volume.
• Mg²⁺ concentrations, buffer pH and cycling conditions in the reaction:  Need high optimization for the amplification reaction.

Recombinant DNA Creation Using DNA Ligase

• Use of maximum ligation mixture: Need to use the recommended amount of the ligation reaction for transformation. (For example: QuickLink™ DNA Ligation Kit and Rapid DNA Ligation Kit can be used for high efficiency and fast ligation.
• Inefficient ligation: Confirm at least one fragment needs to be ligated containing a 5´ phosphate moiety. Purify the DNA to remove contaminants such as salt and EDTA. Remove phosphatase prior to ligation. Confirm the activity of the ligase by carrying out a ligation control may help.
• Inefficient phosphorylation:  Need to purify DNA prior to phosphorylation to remove excess salt, phosphate or ammonium ions that may inhibit the kinase.
• Inefficient blunting: Heat inactivation or removal of the restriction enzymes prior to blunting and cleaning the PCR fragment prior to blunting may help.
• Inefficient A-Tailing: Need to clean the PCR prior to A-tailing. The high-fidelity enzymes will remove any non-templated nucleotides)

The ligated DNA ran as a smear on an agarose gel

• The ligase is bound to the DNA substrate: Need to treat the ligation reaction with Proteinase K (for example: Proteinase K from Tritirachium album) prior to running on a gel.

Insertion of Recombinant DNA into Host Organism

• If cells are not viable: Need to calculate the transformation efficiency of the competent cells or use of commercially available high efficiency competent cells. For example: SIG10 Chemically Competent Cells (CMC0001) possess high transformation efficiency of >1 x 10⁹ cfu/μg
• Gene of interest (GOI) may be toxic to the cells: Need to incubate plates at lower temperature or using a strain that exerts stronger transcriptional control over the DNA fragment or gene of interest. For example: CONTROLLER SIG10 Chemically Competent Cells can be used.
• If the wrong heat-shock protocol is used (for chemically competent cells): Need to follow the manufacturer’s specific transformation protocol and use of recommended temperature during the heat shock will be helpful.
• If PEG is present in the ligation mix (for electrocompetent cells): Needs cleaning of DNA by drop dialysis prior to transformation.
• If no voltage is registered in case of electrocompetent cells: Need to clean DNA prior to the ligation step and remove any air bubbles. Need to follow the manufacturer’s specified electroporation parameters.
• Constructed DNA is too large: Need to select a competent cell strain that can be transformed efficiently with large DNA constructs or use of electroporation may help. For example: XLDNA SIG10 Electrocompetent cells can be used for large constructs and increase in DNA yields.
• Constructing a DNA fragment that may be susceptible to recombination:  Needs to be checked. For example: STEADY Chemically Competent Cells can be used.
• For selection of organisms containing vector sequence, a selectable marker (usually a gene is essential) that confers resistance to an antibiotic.
  Incorrect antibiotic or its concentration: Need to confirm antibiotic and optimize its concentration. The selection marker used for the vector also needs to be confirmed. For example Hygromycin B from Streptomyces hygroscopicus can be used as a marker. Please find the below table with few examples of antibiotics along with their working concentration.

Table 1. Some antibiotics commonly used, their mechanism, and general working concentrations.

Antibiotic Name	Class	Mode of Action*	Type	Working Concentration**
Kanamycin (Product No. K1377)	aminoglycoside	Binds 30S ribosomal subunit; causes mis-translation	Bactericidal	100 mg/L
Spectinomycin (Product No. S4014)	aminoglycoside	Binds 30S ribosomal subunit; interrupts protein synthesis	Bactericidal	7.5-20 mg/L
Ampicillin (Product Nos. A6140, A9393 and A9518)	beta-lactam	Inhibits cell wall synthesis	Bactericidal	20-60 µg/mL
Carbenicillin (Product No. C3416)	beta-lactam	Inhibits cell wall synthesis	Bactericidal	100 µg/mL
Bleomycin (Product No. B5507)	glycopeptide	Induces DNA breaks	Bactericidal	10-100 µg/mL
Erythromycin (Product No. E6376)	macrolide	Blocks 50S ribosomal subunit; inhibits aminoacyl translocation	Bacteriostatic	50-200 mg/L
Polymyxin B (Product No. P1004)	polypeptide	Alters outer membrane permeability	Bactericidal	10-100 µg/mL
Tetracycline (Product No. T3383)	tetracyclin	Binds 30S ribosomal subunit; inhibits protein synthesis (elongation step)	Bacteriostatic	10 mg/mL
Chloramphenicol (Product No. C0378)		Binds 50S ribosomal subunit; inhibits peptidyl translocation	Bacteriostatic	10-20 µg/mL
*In prokaryotes. **Dissolve in ultrapure water and sterile filter unless otherwise specified.

In addition to the possible causes shown in the Troubleshooting Guide, the following problems can also be associated with low or no transformation efficiency and can be checked.

• Impurities in the DNA: Need to remove phenol, proteins, detergents and ethanol from the DNA solution when using chemically competent cells (i.e. BL21(DE3)). Ethanol precipitates during ligations to clean up plasmid DNA when using electrocompetent cells i.e. BL21(DE3)  (since salt and buffers strongly inhibit electroporation and increase the risk of arcing). DNA can also be dissolve in sterile water to check for further impurities.
• Excess DNA or volume: This needs to be checked and only the recommended volume needs to be prepared depending upon the type of cells used.
• Low or poor expression of antibiotic resistance: S.O.C. medium is recommended for expression instead of LB medium as it decreases the transformation efficiency by 2-3 folds.
• Improper storage and handling of competent cells: Store competent cells at -80 °C and not in liquid nitrogen. Competent cells should be used as soon as they are thawed and remain cold on ice until use. Minimize the number of freeze-thaw cycles.
• Slow or no growth of cells or over growth: Using the recommended temperature may help in proper growth of cells. For overgrowth, need to be particular about the antibiotic used and its concentration.
• Improper calculations: Ensure the correct dilution factors and DNA amounts are used to calculate efficiency.

Clone Screening

Colonies without a plasmid:

• Insufficient amount of antibiotic used:  Need to increase the antibiotic level on plates to the recommended amount and use of fresh plates with fresh antibiotics may help. Please refer to table 1.
• Satellite colonies selection: Need to select large, well-established colonies for further analysis.
  (Satellite colonies are very small colonies of cells growing around an antibiotic resistant colony and have taken up the wrong plasmid. The antibiotic resistant colony releases enzymes that degrade the antibiotic. The satellites colonies do not grow when transferred to a fresh medium, so they are not considered for further analysis.)

Colonies containing the wrong/incorrect construct may be due to:

• Recombination of the plasmid (vector)
• Incorrect PCR amplicon used during cloning: Need to optimize the PCR conditions. 
• Presence of internal recognition site: Need to analyze insert sequence for the presence of an internal recognition site.
• Presence of mutations/error in the sequence:
  • Use of low fidelity DNA polymerase: Use a high-fidelity DNA polymerase with Proofreading activity for example: Further re-running the sequencing reaction can be helpful.
  • DNA damaged by UV light during the excision from gel: Use of long wavelength UV (360 nm) light-box is recommended while excising DNA from the agarose gel.

Consideration for PCR Cloning

• Nature of the insert or fragment used: The efficiency of the PCR fragment differs due to fragment size, insert toxicity, and the complexity of the insert.
• Insert Size: Small fragments of DNA are considered for high efficiency.
• Vector-to-Insert Ratio:  It is very essential and critical to optimize the molar concentration ratios of the vector to insert for efficient cloning.
• Use of clean PCR Product: It is recommended to use clean PCR product and for some cases use of fresh product is helpful. (For example: rAPid Alkaline Phosphatase can be used for clean-up of PCR product by removing dNTPs).
• Use of Positive and Negative Controls: It is essential to have an appropriate positive and negative control for evaluating the results of a cloning event.
• Compatibility of DNA Ends of Vector and Insert: This needs to be checked and for successful cloning the use of correct polymerase with the cloning vector is important. Use of SnapFast™ vector makes cloning efficient and easy as it makes moving DNA from one vector to another easy. It is compatible with many pre-existing cloning vectors and a range of shuttle vectors to facilitate gene transfer with high success rates.
• Proper design of forward and reverse PCR Primers as well as probes is crucial.³

References

1. Sharma K, Mishra AK, Mehraj V, Duraisamy GS. 2014. Advances and applications of molecular cloning in clinical microbiology. Biotechnology and Genetic Engineering Reviews. 30(1):65-78. https://doi.org/10.1080/02648725.2014.921501

2. Roux KH. 2009. Optimization and Troubleshooting in PCR. Cold Spring Harbor Protocols. 2009(4):pdb.ip66-pdb.ip66. https://doi.org/10.1101/pdb.ip66

3. Raso A, Mascelli S, Nozza P, Ugolotti E, Vanni I, Capra V, Biassoni R. 2011. Troubleshooting fine-tuning procedures for qPCR system design. J. Clin. Lab. Anal.. 25(6):389-394. https://doi.org/10.1002/jcla.20489

4. Canene-Adams K. 2013. Explanatory Chapter.271-278. https://doi.org/10.1016/b978-0-12-418687-3.00022-7

5. Lorenz TC. Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies. JoVE.(63): https://doi.org/10.3791/3998

6. Fulton TL, Stiller M. 2012. PCR Amplification, Cloning, and Sequencing of Ancient DNA.111-119. https://doi.org/10.1007/978-1-61779-516-9_15

7. Mergulhão F, Kelly A, Monteiro G, Taipa MA, Cabral JM. Troubleshooting in Gene Splicing by Overlap Extension: A Step-Wise Method. MB. 12(3):285-288. https://doi.org/10.1385/mb:12:3:285

Please contact our technical support if your case cloning problem persists.