Molecular Cloning Screening and Identification Technologies: Positive Clone Confirmation, Restriction Mapping, and Sequencing Validation

Molecular cloning screening and identification are used to confirm whether the target insert has been correctly introduced into the vector, whether the insertion orientation matches the design, whether the reading frame is continuous, whether sequence mutations are present, and whether the construct is reliable for downstream expression, transfection, functional validation, or plasmid storage. Common methods include antibiotic resistance screening, blue-white screening, colony PCR, restriction enzyme digestion, plasmid sequencing, and downstream functional validation.

Keywords: molecular cloning screening; positive clone identification; colony PCR; restriction digestion identification; blue-white screening; plasmid sequencing; vector construction

1 Basic Logic of Molecular Cloning Screening and Identification

1.1 Key Questions to Confirm During Screening

(1) Whether transformants have been obtained

Colonies growing on antibiotic plates only indicate that cells have acquired a DNA molecule carrying an antibiotic resistance marker. This does not directly prove that the target insert has been correctly inserted. Empty vectors, self-ligated vectors, or incorrectly assembled products may also form colonies. Therefore, antibiotic resistance screening can only serve as an initial screening step for candidate clones.

(2) Whether the target insert is present

Colony PCR, insert-specific PCR, and restriction digestion can be used to determine whether candidate clones contain the target insert. This level mainly confirms the presence or absence of the insert, but does not fully determine insertion orientation, reading-frame continuity, or full sequence correctness.

(3) Whether the insertion orientation and junction boundaries are correct

Expression vectors, promoter-driven vectors, fusion tag vectors, and reporter gene vectors require particular confirmation of insert orientation and junction boundaries. Vector primers combined with insert primers, double-digestion patterns, and junction sequencing can be used to determine whether the construct matches the design.

(4) Whether the sequence is completely correct

Point mutations, deletions, or rearrangements may be introduced during PCR amplification, ligation, recombination, or bacterial propagation. Clones intended for expression, transfection, functional analysis, or long-term storage should be verified by sequencing that covers the insert, junction regions, and key regulatory elements.

1.2 Common Screening Workflows

(1) Rapid preliminary screening workflow

After transformation, candidate colonies are first obtained on antibiotic plates and then subjected to colony PCR to rapidly exclude empty vectors and obviously incorrect clones. This workflow is suitable for routine single-insert cloning, TA cloning, and conventional vector construction.

(2) Structural confirmation workflow

Colony PCR-positive clones are further processed by plasmid miniprep and restriction digestion. Restriction patterns can be used to evaluate the vector backbone, insert size, and partial orientation, and are suitable for expression vectors, large-insert cloning, and multi-fragment construction.

(3) Final confirmation workflow

Sanger sequencing is used to confirm junction boundaries, reading frame, mutation sites, and open reading frame integrity. Before functional experiments, the final basis for construct confirmation should be sequencing results rather than a single PCR band or restriction pattern.

Table 1 Application Positioning of Molecular Cloning Screening and Identification Methods

Method	Main Question Addressed	Applicable Stage	Key Interpretation Point
Antibiotic resistance screening	Whether transformants carrying a resistance marker have been obtained	Initial screening after transformation	Cannot distinguish empty vectors from positive clones
Blue-white screening	Preliminary distinction between insertional inactivation and empty vector	TA cloning and lacZ-related vectors	White colonies still require PCR or sequencing confirmation
Colony PCR	Whether the target insert or junction is present	Initial positive clone screening	Primer positions determine the interpretive scope
Plasmid miniprep	Obtaining plasmid DNA for detection and storage	Confirmation after preliminary screening	Concentration, purity, and integrity should be checked
Restriction digestion	Determining insert size and partial orientation	Structural confirmation	A discriminating digestion strategy is required
Sanger sequencing	Confirming sequence, junctions, and reading frame	Final confirmation	Sequencing coverage must be complete
Functional validation	Determining whether the construct can express or produce function	Downstream application	Expression results cannot replace sequence confirmation

2 Preliminary Screening After Transformation

2.1 Antibiotic Resistance Screening

(1) Selection of resistance markers

The antibiotic used must match the resistance marker encoded by the vector. Ampicillin, kanamycin, chloramphenicol, and tetracycline are commonly used in different vector systems. If the wrong antibiotic is selected, there may be no colonies or an abnormal increase in background colonies.

(2) Control setup

A positive transformation control is used to determine whether competent cells and the transformation step are functioning normally. A negative control is used to exclude medium contamination and antibiotic failure. If colonies appear in the negative control, contamination, antibiotic concentration, and medium preparation conditions should be checked first.

(3) Interpretation of colony number

A large number of colonies does not necessarily indicate a high proportion of positive clones. Vector self-ligation, incomplete digestion, or high empty-vector background can also produce many colonies. If the colony number is low, the ligation system, competent cell efficiency, recovery conditions, and insert toxicity should all be evaluated.

2.2 Blue-White Screening

(1) Screening principle

Blue-white screening depends on lacZα complementation. When the target insert disrupts the multiple cloning site within lacZα, colonies are usually white. Empty vectors in which lacZα is not disrupted appear blue in the presence of IPTG and X-Gal.

(2) Interpretation of white colonies

White colonies are only candidate positive clones and do not directly confirm correct insertion of the target fragment. Small inserts, vector mutations, diffusion of color development, prolonged incubation, or accidental reading-frame restoration may all cause deviations in color interpretation.

(3) Colony selection strategy

White single colonies with clear borders, moderate size, and good separation should be prioritized. Half-blue/half-white colonies, merged colonies, or colonies with abnormal morphology should be selected cautiously and further confirmed by PCR and sequencing.

2.3 Colony Quality Control

(1) Single-clone origin

Colonies used for screening should originate from a single transformation event. Merged colonies, mixed colonies, or regions with obvious morphological differences may lead to multiple PCR bands, mixed plasmids, or double peaks in sequencing.

(2) Repeated colony picking

For routine single-insert cloning, 6–12 independent colonies can be screened first. For TA cloning, blunt-end cloning, multi-fragment assembly, or large-insert cloning, the number of candidate clones should be increased to improve the probability of obtaining a correct clone.

(3) Contamination troubleshooting

If colonies appear on a no-DNA control plate, the medium, antibiotics, inoculation tools, and operating environment should be checked first. Before contamination is excluded, results from test clones should not be interpreted further.

3 Positive Clone Identification Technologies

3.1 Colony PCR

(1) Insert-specific PCR

Primers located inside the insert can rapidly determine whether candidate clones contain the target sequence and are suitable for preliminarily excluding empty vectors. However, this method cannot determine insertion orientation, vector junction status, or overall structural integrity.

(2) Vector-insert junction PCR

One primer is located in the vector and the other in the insert, allowing determination of insertion orientation and junction boundaries. Expression vectors, tag-fusion vectors, and promoter-downstream cloning are more suitable for this design.

(3) Interpretation of abnormal bands

Weak bands, multiple bands, or obviously smaller-than-expected bands are commonly caused by excessive template, insufficient colony lysis, nonspecific amplification, mixed colonies, or insert deletion. If necessary, a single colony should be picked again, or PCR confirmation should be performed after plasmid miniprep.

Table 2 Colony PCR Primer Design and Interpretation

Primer Combination	Information Determined	Advantage	Limitation
Internal insert primers	Whether the target sequence is present	Fast and sensitive	Cannot determine vector junction status
Vector primers flanking both sides	Insert size	Can distinguish empty vector from insert-containing vector	Large inserts may amplify inefficiently
Vector forward primer + insert reverse primer	Insertion orientation and one junction	Suitable for directional screening of expression vectors	Only covers one junction
Insert forward primer + vector reverse primer	Opposite orientation or the other junction	Complements the previous primer set	Annealing temperature must be reasonably designed
Tag-region primer + insert primer	Tag fusion reading frame	Suitable for fusion protein vectors	Cannot replace sequencing confirmation

3.2 Plasmid Miniprep and Restriction Digestion Identification

(1) Plasmid miniprep

Colony PCR-positive clones should be cultured in liquid medium and subjected to plasmid miniprep. A260/A280, A260/A230, DNA concentration, and plasmid integrity can affect subsequent restriction digestion, sequencing, and transfection experiments.

(2) Single-enzyme digestion

Single digestion can be used to determine whether the plasmid can be linearized and to estimate whether the overall plasmid size matches expectations. This method is suitable for preliminary confirmation of vector structure but has limited ability to detect insertion orientation and small-fragment abnormalities.

(3) Double-enzyme digestion

Double digestion can release the target insert or generate characteristic bands, making it more suitable for determining insert size, vector backbone, and partial orientation. If the target insert and vector fragment sizes are close, the digestion combination should be redesigned or confirmed by sequencing.

3.3 Sanger Sequencing Confirmation

(1) Junction sequencing

Sequencing should cover both junctions between the vector and the insert. Correct junctions confirm whether the ligation site, orientation, reading frame, and tag fusion match the design.

(2) Open reading frame sequencing

Expression clones should be sequenced across the complete ORF, especially PCR-amplified regions, mutation-introduced regions, restriction sites, and homologous recombination junctions. Sequencing only one end is usually insufficient to confirm the correctness of a long insert.

(3) Handling abnormal chromatograms

Double peaks, local mixed peaks, sudden read-length decline, or frameshift-like chromatograms are commonly caused by mixed clones, repetitive sequences, secondary structures, or poor template quality. A single clone can be purified by streaking again, or sequencing primers can be redesigned for reverse confirmation.

Table 3 Confirmation Levels for Molecular Cloning Identification

Confirmation Level	Representative Method	What It Can Confirm	What It Cannot Replace
Preliminary screening	Antibiotic resistance screening, blue-white screening	Whether candidate clones exist	Insert and sequence correctness
Insert confirmation	Colony PCR	Whether the target insert is present and partial orientation	Complete sequence and mutation status
Structural confirmation	Restriction digestion	Fragment size, vector structure, partial orientation	Single-base mutations and small deletions
Sequence confirmation	Sanger sequencing	Junctions, reading frame, mutations, and sequence integrity	Protein expression and functional activity
Functional confirmation	Expression detection, reporter gene assay, enzyme activity	Downstream functional usability	Correctness of the DNA construct sequence

4 Screening Points for Different Cloning Strategies

4.1 Restriction Digestion-Ligation Cloning

(1) Double-digestion ligation

Double digestion reduces vector self-ligation and provides directionality. Screening should focus on insert size and orientation, commonly using colony PCR, double digestion to release the insert, and sequencing confirmation.

(2) Single-digestion ligation

Single-digestion ligation is prone to vector self-ligation and bidirectional insertion. More candidate colonies should be screened, and directional PCR or a discriminating restriction pattern should be used to determine orientation.

(3) Dephosphorylation treatment

Vector dephosphorylation can reduce empty-vector background, but excessive treatment may lower ligation efficiency. If colony number is low, vector recovery amount, insert-to-vector molar ratio, and ligase activity should also be checked.

4.2 TA Cloning and Blunt-End Cloning

(1) TA cloning

TA cloning is suitable for rapid insertion of PCR products and is often combined with blue-white screening. White colony candidates still require colony PCR confirmation of insert size and sequencing to exclude PCR-introduced mutations.

(2) Blunt-end cloning

Blunt-end ligation has uncontrolled orientation and usually lower ligation efficiency than sticky-end ligation. More candidate clones should be screened, and vector-insert junction PCR should be used to determine orientation.

(3) PCR product quality

Primer dimers, nonspecific bands, or residual salts in PCR products can reduce ligation efficiency. PCR products should be purified or gel-extracted before ligation, and inserts intended for expression should preferably be amplified using a high-fidelity polymerase.

4.3 Gibson Assembly and Homologous Recombination Cloning

(1) Homology arm design

Gibson Assembly depends on homologous overlapping regions between fragments. Common screening errors include fragment loss, incorrect assembly, and recombination of repetitive sequences. Each junction should be confirmed by junction-spanning PCR and sequencing.

(2) Multi-fragment assembly

The more fragments included, the higher the probability of incorrect assembly. Multi-fragment constructs cannot be judged correct using only one PCR assay; sequencing primers should be designed for each junction.

(3) Large-fragment cloning

Large fragments are more prone to rearrangement, deletion, and low transformation efficiency. Stable host strains, shortened culture time, and restriction mapping, multi-primer sequencing, or long-read sequencing can be used for confirmation.

Table 4 Screening and Identification Priorities for Different Cloning Strategies

Cloning Strategy	Main Risk	Recommended Screening Method	Final Confirmation Method
Double-digestion ligation	Insert loss, incomplete digestion	Colony PCR, double digestion	Bidirectional or full-length sequencing
Single-digestion ligation	Empty vector, self-ligation, reverse insertion	Directional PCR, restriction digestion	Sequencing of orientation and junctions
TA cloning	False white colonies, PCR mutations	Blue-white screening, colony PCR	Insert sequencing
Blunt-end cloning	Low ligation efficiency, random orientation	Multi-colony PCR, directional PCR	Sequencing confirmation
Gibson Assembly	Incorrect assembly, fragment loss	Junction-spanning PCR	Sequencing of multiple junctions
Multi-fragment assembly	Incorrect fragment order, partial deletion	Segmental PCR, restriction mapping	Sequencing covering all junctions
Large-fragment cloning	Rearrangement, deletion, low stability	Restriction mapping, low-copy screening	Long-read or multi-primer sequencing

5 Common Abnormal Results and Optimization Strategies

5.1 No Colonies or Too Few Colonies

(1) Insufficient transformation efficiency

If the positive control also shows no colonies, competent cells, recovery time, heat-shock or electroporation conditions should be checked first. This type of problem should not be directly attributed to ligation failure.

(2) Abnormal ligation system

Improper insert-to-vector ratio, incompatible DNA ends, inactive ligase, residual salts, or incomplete digestion can all reduce ligation efficiency. Vector self-ligation controls and insert quality checks can help locate the issue.

(3) Insert toxicity

Some genes are toxic to the host or affect plasmid stability. A low-copy vector, repressor-containing host, low-temperature culture, or shortened culture time may be attempted.

5.2 Excessive Background Colonies

(1) Vector self-ligation

Single-digested vectors, incomplete digestion, or insufficient dephosphorylation can easily lead to empty-vector background. Double digestion, gel extraction of the correct band, and vector dephosphorylation can reduce this problem.

(2) Antibiotic failure

If colonies appear on negative control plates, antibiotic concentration, storage conditions, and medium cooling temperature should be checked. Antibiotic failure makes background colonies uninterpretable.

(3) False positives in blue-white screening

White colonies do not necessarily represent correct insertion of the target fragment. Pale-colored, half-blue/half-white, or poorly defined colonies should be selected cautiously and must be confirmed by PCR and sequencing.

5.3 PCR Positive but Sequencing Incorrect

(1) PCR-introduced mutations

High-fidelity polymerases can reduce mutation rates but cannot completely eliminate errors. Expression clones and mutant clones should be sequenced across the full ORF rather than only at junctions.

(2) Mixed clones

If colonies are not purified or a mixed colony is picked, sequencing chromatograms may show double peaks. Single colonies should be isolated again by streaking, followed by plasmid miniprep and sequencing.

(3) Repetitive sequences or GC-rich regions

Repetitive sequences, strong secondary structures, and GC-rich regions can lead to reduced sequencing read length or local abnormal chromatograms. Sequencing primers can be changed, sequencing direction adjusted, or segmental sequencing used.

Table 5 Abnormal Results in Molecular Cloning Screening and Recommended Handling Strategies

Abnormal Result	Possible Cause	Priority Handling Strategy
No colonies after transformation	Competent cell failure, ligation failure, wrong antibiotic	Check positive control and resistance marker
Many colonies but PCR negative	High empty-vector background, vector self-ligation	Optimize digestion, dephosphorylation, and insert ratio
Multiple bands in colony PCR	Excessive template, nonspecific amplification, mixed colonies	Reduce template amount and pick a single colony again
Correct PCR band but abnormal digestion	Rearrangement, partial deletion, mixed plasmid	Repeat miniprep and confirm by sequencing
Correct digestion but sequencing mutation	PCR amplification error, replication mutation	Screen multiple independent clones again
Obvious double peaks in sequencing	Mixed clone, impure plasmid template	Purify by streaking and sequence again
Expression failure despite correct sequence	Tag, reading frame, toxicity, or folding issue	Check expression vector design and host system

6 Products Related to Molecular Cloning Screening and Identification

Table 6 Reagents, Enzymes, and Detection Materials Related to Molecular Cloning Screening and Identification

Cat. No.	Product Name	Grade/Specification	Product Category	Application Positioning
T665705	T4 DNA Ligase	Bioactive,Recombinant,ActiBioPure™,High Performance,EnzymoPure™,1000 U/μl	DNA ligase	Used for sticky-end ligation, blunt-end ligation, and routine vector-insert construction
T397964	T4 DNA Ligase (Fast)	EnzymoPure™	DNA ligase	Used for molecular cloning ligation reactions, positive clone construction, and vector self-ligation controls
T292950	Taq DNA Ligase	EnzymoPure™, 40U/μl	Thermostable DNA ligase	Used for oligonucleotide ligation, ligation detection, and ligase-dependent identification systems
E745500	E. coli DNA Ligase	Bioactive,Recombinant,ActiBioPure™,High Performance,EnzymoPure™,10 U/μl	DNA ligase	Used for DNA nick ligation, ligation mechanism research, and optimization of specific cloning systems
P748845	PBCV-1 DNA Ligase	Bioactive,Recombinant,ActiBioPure™,High Performance,EnzymoPure™,25 U/μl	DNA ligase	Used for special end ligation, ligation system comparison, and in vitro DNA assembly research
T750867	T3 DNA Ligase	Bioactive,Recombinant,ActiBioPure™,High Performance,EnzymoPure™,3 KU/μl	DNA ligase	Used for DNA ligation reactions, ligase system comparison, and fragment ligation optimization
T750877	T7 DNA Ligase	Bioactive,Recombinant,ActiBioPure™,High Performance,EnzymoPure™,3 KU/μl;expressed in E.coli	DNA ligase	Used for DNA ligation reactions, ligation after end repair, and ligation efficiency comparison
FP1509106	HiFi Seq Hotstart DNA Polymerase	Recombinant,Suitable for molecular biology,EnzymoPure™,for DNA and RNA applications,1 U/μL	High-fidelity hot-start polymerase	Used for expression cloning, mutant construction, and insert amplification requiring reduced PCR mutation rates
H292966	HiFi Seq Hotstart DNA polymerase	1U/μL	High-fidelity DNA polymerase	Used for high-fidelity amplification of target fragments, sequencing template preparation, and pre-cloning PCR
P292987	Pfu DNA Polymerase	EnzymoPure™, 2.5U/μl	High-fidelity DNA polymerase	Used for low-mutation amplification, blunt-end cloning, and expression vector construction
FP1508912	Taq DNA Polymerase	Recombinant,Suitable for molecular biology,EnzymoPure™,for DNA and RNA applications,5 U/μL	Conventional PCR polymerase	Used for colony PCR, positive clone preliminary screening, and TA cloning insert amplification
T665586	Taq DNA Polymerase	Recombinant,Suitable for molecular biology,EnzymoPure™,for DNA and RNA applications,≥99%(SDS-PAGE),5 U/μl	Conventional PCR polymerase	Used for colony PCR, insert detection, and rapid transformant screening
FP1508958	Epitech Taq DNA Polymerase	Recombinant,Suitable for molecular biology,EnzymoPure™,for DNA and RNA applications,5 U/μL	Conventional PCR polymerase	Used for positive clone PCR screening and routine fragment amplification
FP1508941	Epitech HS Taq DNA Polymerase	Recombinant,Suitable for molecular biology,EnzymoPure™,for DNA and RNA applications,5 U/μL	Hot-start PCR polymerase	Used to improve colony PCR specificity and reduce nonspecific amplification
FP1509104	Hotstart HiTaq DNA Polymerase	Recombinant,Suitable for molecular biology,EnzymoPure™,for DNA and RNA applications,5 U/μL	Hot-start PCR polymerase	Used for vector-insert junction PCR and directional clone screening
FP1508913	Hotstart HiTaq Ⅱ DNA Polymerase	Suitable for molecular biology,EnzymoPure™,for DNA and RNA applications,5 U/μL	Hot-start PCR polymerase	Used for complex-template colony PCR and positive clone preliminary screening
H292167	Hot Start Taq DNA Polymerase	EnzymoPure™, 5U/μL	Hot-start PCR polymerase	Used to improve PCR identification specificity and confirm weak positive clones
T1424927	Taq DNA Polymerase, Glycerol-free		Hot-start PCR polymerase	Used for PCR amplification and clone identification in glycerol-sensitive systems
FP1508925	AK Taq DNA Polymerase V2	Suitable for molecular biology,EnzymoPure™,for DNA and RNA applications,5 U/μL	PCR polymerase	Used for routine PCR, colony PCR, and cloning fragment amplification
FP1508914	ProPrime Taq DNA Polymerase	Suitable for molecular biology,EnzymoPure™,for DNA and RNA applications,5 U/μL	PCR polymerase	Used for positive clone preliminary screening and insert amplification
FP1509109	PowerResist Taq Polymerase	Suitable for molecular biology,EnzymoPure™,for DNA and RNA applications,5 U/μL	PCR polymerase	Used for complex-template PCR and cloning screening amplification
FP1509151	Rock DNA Polymerase	EnzymoPure™,Suitable for molecular biology,2.5 U/μL	PCR polymerase	Used for routine fragment amplification, colony PCR, and cloning identification
F1449950	FastTaq DNA Polymerase(5'→3' exo-)		PCR polymerase	Used for rapid PCR amplification and positive clone preliminary screening
G1444565	Golden Taq DNA Polymerase		PCR polymerase	Used for routine PCR amplification and colony PCR identification
T295115	Taq-HS DNA Polymerase	EnzymoPure™	Hot-start DNA polymerase	Used for high-specificity PCR, complex-template amplification, and directional identification
A295123	AbTaq DNA Polymerase	EnzymoPure™	Hot-start DNA polymerase	Used to reduce nonspecific amplification and improve colony PCR interpretation accuracy
T295106	Taq DNA Polymerase	EnzymoPure™	Thermostable DNA polymerase	Used for high-temperature PCR amplification and cloning fragment amplification
W292915	Whole Blood polymerase	EnzymoPure™, 1.25U/μl	DNA polymerase	Used for specific PCR amplification and cloning fragment preparation
FP1508928	T4 DNA Polymerase	Suitable for molecular biology,EnzymoPure™,for DNA and RNA applications,3 U/μL	End-repair enzyme	Used for DNA end polishing, blunt-end cloning, and vector end treatment
T665886	T4 DNA Polymerase	EnzymoPure™, Animal Free, Carrier Free, Bioactive, ActiBioPure™, sterile, RNase free, 5.0 U/μL	End-repair enzyme	Used for DNA end repair, blunt-end ligation, and cloning fragment treatment
L398126	ApaLI	EnzymoPure™	Restriction endonuclease	Used for vector digestion, insert release, and plasmid mapping identification
L354062	AscI	EnzymoPure™	Restriction endonuclease	Used for large-fragment cloning, rare-cutter linearization, and recombinant plasmid identification
L358929	AvrII	EnzymoPure™	Restriction endonuclease	Used for directional cloning and restriction mapping confirmation
L358964	BamHI	EnzymoPure™	Restriction endonuclease	Used for vector double digestion, insert release, and digestion-based identification
L359021	BclI	EnzymoPure™	Restriction endonuclease	Used for site-specific digestion and cloning structure analysis
L398083	BglII	EnzymoPure™	Restriction endonuclease	Used for clone fragment release, vector construction, and double-digestion identification combinations
L398114	BstBI	EnzymoPure™	Restriction endonuclease	Used for vector linearization and plasmid structure identification
L359061	BstEII	EnzymoPure™	Restriction endonuclease	Used for site-specific digestion and recombinant plasmid mapping analysis
L359076	ClaI	EnzymoPure™	Restriction endonuclease	Used for directional cloning and double-digestion identification
L397155	DpnII	EnzymoPure™	Restriction endonuclease	Used for GATC-site-related digestion analysis and plasmid structure interpretation
L397162	EagI	EnzymoPure™	Restriction endonuclease	Used for recombinant plasmid linearization and low-frequency cutter identification
L397257	EcoRI	EnzymoPure™	Restriction endonuclease	Used for routine cloning site digestion, vector linearization, and recombinant plasmid identification
L397409	EcoRV	EnzymoPure™	Restriction endonuclease	Used for blunt-end digestion, vector linearization, and fragment structure confirmation
L397895	FspI	EnzymoPure™	Restriction endonuclease	Used for plasmid linearization and restriction mapping analysis
L398116	HinfI	EnzymoPure™	Restriction endonuclease	Used for restriction fragment analysis and small-fragment pattern interpretation
L397431	HpaI	EnzymoPure™	Restriction endonuclease	Used for blunt-end digestion, structural verification, and vector linearization
L397446	KpnI	EnzymoPure™	Restriction endonuclease	Used for directional cloning, double-digestion identification, and insert release
L397459	MluI	EnzymoPure™	Restriction endonuclease	Used for expression vector construction, pre-treatment before multi-fragment assembly, and directional identification
L397466	MnlI	EnzymoPure™	Restriction endonuclease	Used for restriction fragment analysis and detailed cloning structure identification
L397482	NcoI	EnzymoPure™	Restriction endonuclease	Used for expression cloning around the start codon region and reading-frame confirmation
L397493	NdeI	EnzymoPure™	Restriction endonuclease	Used for expression vector start-region cloning and ORF insertion orientation confirmation
L397598	NheI	EnzymoPure™	Restriction endonuclease	Used for expression vector construction, tag-fusion boundary identification, and double-digestion analysis
L397626	NotI	EnzymoPure™	Restriction endonuclease	Used for large-insert cloning, low-frequency cutter identification, and vector structure confirmation
L398105	NsiI	EnzymoPure™	Restriction endonuclease	Used for site-specific digestion and plasmid mapping identification
L398140	PacI	EnzymoPure™	Restriction endonuclease	Used for large-fragment cloning, rare-cutter plasmid identification, and vector linearization
L397656	PstI	EnzymoPure™	Restriction endonuclease	Used for multiple cloning site digestion, insert release, and plasmid mapping verification
L398090	PvuII	EnzymoPure™	Restriction endonuclease	Used for blunt-end digestion, vector linearization, and restriction mapping identification
L397676	SacI	EnzymoPure™	Restriction endonuclease	Used for insert release, vector backbone confirmation, and double-digestion identification
L398098	SacII	EnzymoPure™	Restriction endonuclease	Used for site-specific digestion identification and complex vector structure analysis
L397693	SbfI	EnzymoPure™	Restriction endonuclease	Used for large-fragment cloning, rare-cutter digestion, and structural confirmation
L397704	SmaI	EnzymoPure™	Restriction endonuclease	Used for blunt-end cloning, vector linearization, and digestion verification
L397720	Spel	EnzymoPure™	Restriction endonuclease	Used for multiple cloning site construction, directional insertion, and junction verification
L397780	SphI	EnzymoPure™	Restriction endonuclease	Used for insert release and specific multiple cloning site identification
L397795	SspI	EnzymoPure™	Restriction endonuclease	Used for plasmid linearization and blunt-end digestion analysis
L397810	StuI	EnzymoPure™	Restriction endonuclease	Used for blunt-end digestion, structural verification, and plasmid mapping analysis
L397827	TaqI	EnzymoPure™	Restriction endonuclease	Used for site-specific restriction analysis and cloning pattern interpretation
L397840	XbaI	EnzymoPure™	Restriction endonuclease	Used for directional cloning, expression vector construction, and recombinant plasmid identification
L397875	XhoI	EnzymoPure™	Restriction endonuclease	Used for expression vector double digestion, ORF insertion, and restriction pattern confirmation
A397978	Alkaline Phosphatase (Fast)	EnzymoPure™	Dephosphorylation enzyme	Used for dephosphorylating digested vectors to reduce vector self-ligation background
S1438762	Shrimp Alkaline Phosphatase		Dephosphorylation enzyme	Used for vector-end dephosphorylation and pre-ligation treatment
rp180222	Alkaline Phosphatase (ALP)	EnzymoPure™, ≥5000 U/mg	Dephosphorylation enzyme	Used for DNA-end dephosphorylation, vector self-ligation control, and cloning system optimization
A755483	Alkaline Phosphatase	EIA grade, from calf intestine	Dephosphorylation enzyme	Used for vector-end dephosphorylation and ligation background control
P128629	Phosphatase, Alkaline	EnzymoPure™, Native, ≥30 units/mg protein (25°C, pH 8.0),from Escherichia coli	Dephosphorylation enzyme	Used for vector-end treatment and cloning system optimization
P755447	Alkaline Phosphatase from calf intestinal	Bioactive,ActiBioPure™,Native,High Performance,EnzymoPure™,≥97%(HPLC),≥5000 U/mg protein; Protein concentration: 10-15mg/mL	Dephosphorylation enzyme	Used for efficient dephosphorylation to reduce vector self-ligation
D598085	DL10000 DNA Marker	0.09 mg/mL	DNA molecular weight standard	Used for judging larger PCR products, digestion fragments, and plasmid linearization bands
B598088	DL15000 DNA Marker	0.064mg/mL	DNA molecular weight standard	Used for large inserts, vector backbones, and plasmid digestion pattern analysis
S665584	Super DNA Marker		DNA molecular weight standard	Used for routine PCR, colony PCR, and digestion band-size determination
B598084	100bp DNA Ladder		DNA molecular weight standard	Used for short-fragment PCR, insert preliminary screening, and small-fragment cloning identification
D745352	DNA Ladder (50-500bp)	BioReagent, ready-to-use, suitable for electrophoresis, Suitable for molecular biology, for NA electrophoresis, 50-500bp, 9 bands, Blue,  50bp/ 100bp/ 150bp/ 200bp/ 250bp/ 300bp/ 350bp/ 400bp/ 500bp	DNA molecular weight standard	Used for short inserts, small-fragment PCR, and PCR interpretation of blue-white colony candidates
D665544	DNA Ladder (100-2000bp)	BioReagent, ready-to-use, suitable for electrophoresis, Suitable for molecular biology, for NA electrophoresis, 100-2000bp, 6 bands, Blue, 100bp/ 250bp/ 500bp/ 750bp/ 1000bp/ 2000bp	DNA molecular weight standard	Used for routine positive clone PCR and short-to-medium insert identification
D745353	DNA Ladder (100-15000bp)	BioReagent, ready-to-use, suitable for electrophoresis, Suitable for molecular biology, for NA electrophoresis, 100-15000bp, 11 bands, Blue, 100bp/ 250bp/ 500bp/ 750bp/ 1000bp/ 1500bp/ 2000bp/ 3000bp/ 5000bp/ 8000bp/ 15000bp	DNA molecular weight standard	Used for plasmid digestion identification, large inserts, and vector backbone analysis
R751624	Gelred-prestained DNA Ladder (100-1500bp)	BioReagent, ready-to-use, suitable for electrophoresis, Suitable for molecular biology, for NA electrophoresis, 100-1500bp, 11 bands, 100bp/ 200bp/ 300bp/ 400bp/ 500bp/ 600bp/ 700bp/ 800bp/ 900bp/ 1000bp/ 1500bp	Prestained DNA molecular weight standard	Used for rapid interpretation of colony PCR and short-insert results, reducing additional staining steps
R751622	Gelred-prestained DNA Ladder (100-5000bp)	BioReagent, ready-to-use, suitable for electrophoresis, Suitable for molecular biology, for NA electrophoresis, 100-5000bp, 9 bands, 100bp/ 250bp/ 500bp/ 750bp/ 1000bp/ 1500bp/ 2000bp/ 3000bp/ 5000bp	Prestained DNA molecular weight standard	Used for routine PCR, digestion identification, and medium-length insert analysis
G751634	Gelred-prestained DNA Ladder (100-2000bp)	BioReagent, ready-to-use, suitable for electrophoresis, Suitable for molecular biology, for NA electrophoresis, 100-2000bp, 6 bands, 100bp/ 250bp/ 500bp/ 750bp/ 1000bp/ 2000bp	Prestained DNA molecular weight standard	Used for colony PCR, short-fragment clone screening, and rapid electrophoresis interpretation
G751633	Gelred-prestained DNA Ladder (50-500bp)	BioReagent, ready-to-use, suitable for electrophoresis, Suitable for molecular biology, for NA electrophoresis, 50-500 bp, 9 bands, 50bp/ 100bp/ 150bp/ 200bp/ 250bp/ 300bp/ 350bp/ 400bp/ 500 bp	Prestained DNA molecular weight standard	Used for small inserts, primer-amplified fragments, and short PCR product identification
R751623	Gelred-prestained DNA Ladder (250-10000bp)	BioReagent, ready-to-use, suitable for electrophoresis, Suitable for molecular biology, for NA electrophoresis, 250-10000bp, 9 bands, 250bp/ 500bp/ 750bp/ 1000bp/ 1500bp/ 2000bp/ 3000bp/ 5000bp/ 10000bp	Prestained DNA molecular weight standard	Used for plasmid digestion patterns, large inserts, and vector linearization assessment
R751625	Gelred-prestained DNA Ladder (250-12000bp)	BioReagent, ready-to-use, suitable for electrophoresis, Suitable for molecular biology, for NA electrophoresis, 250-12000bp, 13 bands, 250bp/ 500bp/ 750bp/ 1000bp/ 1500bp/ 2000bp/ 2500bp/ 3000bp/ 4000bp/ 5000bp/ 6000bp/ 8000bp/ 12000bp	Prestained DNA molecular weight standard	Used for large-insert cloning, complex digestion patterns, and vector structure identification
D1521308	DM-25bp DNA Marker	Suitable for molecular biology, BioReagent, ready-to-use, for NA electrophoresis, for PAGE, 25-600bp, 11 bands, 25bp/ 50bp/ 75bp/ 100bp (Reference Band)/ 150bp/ 200bp/ 250bp/ 300bp (Reference Band)/ 400bp/ 500bp/ 600bp	Small-fragment DNA marker	Used for very short inserts, primer-dimer discrimination, and small PCR product analysis
D1521306	DM1500 DNA Marker	Suitable for molecular biology, BioReagent, ready-to-use, for NA electrophoresis, for PAGE, 100-1500bp, 11 bands, 100bp/ 200bp/ 300bp/ 400bp/ 500bp (Reference Band)/ 600bp/ 700bp/ 800bp/ 900bp/ 1000bp/ 1500bp	DNA marker	Used for short-fragment PCR, colony PCR, and small insert screening
D1521305	DM2000 DNA Marker	Suitable for molecular biology, BioReagent, ready-to-use, for NA electrophoresis, for PAGE, 100-2000bp, 7 bands, 100bp/ 250bp/ 500bp/ 750bp (Reference Band)/ 1000bp/ 1500bp/ 2000bp	DNA marker	Used for routine positive clone PCR and short-to-medium fragment identification
D1521303	DM5000 DNA Marker	Suitable for molecular biology, BioReagent, ready-to-use, for NA electrophoresis, for PAGE, 100-5000bp, 9 bands, 100bp/ 250bp/ 500bp/ 750bp (Reference Band)/ 1000bp/ 1500bp/ 2000bp/ 3000bp/ 5000bp	DNA marker	Used for medium-length inserts, digestion fragments, and routine plasmid identification
D1521299	DM-20Kb DNA Marker	Suitable for molecular biology, BioReagent, ready-to-use, for NA electrophoresis, for PAGE, 500-20000bp, 10 bands, 500bp/ 1000bp/ 2000bp/ 3000bp/ 4000bp (Reference Band)/ 5000bp/ 6000bp/ 8000bp/ 10000bp/ 20000bp	Large-fragment DNA marker	Used for large-fragment cloning, vector backbones, and long-fragment digestion pattern assessment
R751626	UltraBio™ Red Fluorescent DNA Marker Dye	BioReagent, suitable for electrophoresis, Suitable for molecular biology, for NA electrophoresis, 50X	DNA marker dye	Used for prestaining DNA markers or assisting loading observation during nucleic acid electrophoresis
G751635	UltraBio™ Green Fluorescent DNA Marker Dye	BioReagent, suitable for electrophoresis, Suitable for molecular biology, for NA electrophoresis, 50×	DNA marker dye	Used for DNA marker staining and band visualization during nucleic acid electrophoresis
A118881	Agarose	High resolution, DNase, RNase, NICKase, none detected	Electrophoresis gel material	Used for resolving PCR products, colony PCR bands, and small insert fragments
O434540	OmniPur® Agarose PCR Plus	Agarose suitable for a wide range of nucleic acid and protein gel applications,including resolution of PCR products and small DNA fragments of less than 1000 bp.	Electrophoresis gel material	Used for small-fragment PCR identification, short insert resolution, and preliminary clone screening
O434539	OmniPur® Agarose	Agarose suitable for a wide range of nucleic acid and protein gel applications,including resolution of PCR products and small DNA fragments of less than 1000 bp.	Electrophoresis gel material	Used for routine PCR products, electrophoretic identification, and digestion fragment analysis
O434545	OmniPur® Agarose Super-Fine Resolution		High-resolution agarose	Used for small inserts, short PCR products, and fine band resolution
A104063	Agarose	low melting point ，for seperation of small nucleic fragment	Gel recovery electrophoresis material	Used for low-temperature recovery of digestion fragments and PCR inserts
A434532	Agarose	Suitable for molecular biology, Ultra-low Gelling Temperature	Low-melting-point agarose	Used for gel recovery, fragment purification, and pre-ligation DNA recovery
A434541	Agarose	BioReagent, Suitable for molecular biology, low EEO	Electrophoresis gel material	Used for routine PCR, digestion identification, and plasmid electrophoresis analysis
A434543	Agarose	Suitable for molecular biology, Medium EEO	Electrophoresis gel material	Used for routine nucleic acid electrophoresis and cloning screen band analysis
A434537	Agarose	For pulsed field electrophoresis running gel	Large-fragment electrophoresis material	Used for large DNA fragments, complex plasmids, and long-fragment structure analysis
A397944	Agarose		Electrophoresis gel material	Used for high-quality nucleic acid electrophoresis and clone identification
A501160	Agar	Suitable for molecular biology	Plate culture material	Used for antibiotic plates, transformant screening, and blue-white screening plate preparation
A501173	Agar	Suitable for molecular biology, for bacteria and yeast culture	Plate culture material	Used for E. coli transformant culture and clone screening
A501167	Agar	Suitable for molecular biology, for NZYM agar base	Plate culture material	Used for bacterial medium preparation and transformant screening
A501165	Agar	Suitable for molecular biology, for NZCYM agar base	Plate culture material	Used for bacterial medium preparation and clone culture
E119045	Ethidium bromide(EB)	Suitable for molecular biology, ≥95%(HPLC), powder	Nucleic acid dye	Used for agarose gel DNA visualization and PCR/digestion band observation
E274237	Ethidium bromide	0.625 mg/mLin H2O,BIOTECHNOLOGY GRADE	Nucleic acid dye solution	Used for DNA gel staining and cloning identification electrophoresis detection
E421031	Ethidium bromide(EB)	10mM in DMSO	Nucleic acid dye solution	Used for nucleic acid electrophoresis staining and DNA band observation
E109020	Ethidium bromide(EB)	≥95%(HPLC)	Nucleic acid dye	Used for electrophoretic visualization of PCR products and digestion fragments
R665518	RNase A	EnzymoPure™, DNase free, Protease Free, sterile, ≥90%(SDS-PAGE), 10 mg/mL	RNase A	Used in plasmid miniprep to remove RNA contamination and improve sequencing and digestion template quality
R128619	Ribonuclease A from bovine pancreas(DNase & Protease Free)	Bioactive,ActiBioPure™,Native,High Performance,EnzymoPure™,DNase free,Protease Free,≥2,000 units/mg protein	RNase A	Used for plasmid extraction, DNA purification, and RNA removal from nucleic acid samples
R128622	Ribonuclease A from bovine pancreas	EnzymoPure™, ≥2,500 units/mg dry weight	RNase A	Used for RNA degradation in plasmid extraction systems
R665521	Recombinant RNase A	DNase free, EnzymoPure™, Protease Free, ActiBioPure™, Bioactive, High Performance, Recombinant, ≥90%(SDS-PAGE), 100 mg/mL	Recombinant RNase A	Used for DNA purification, plasmid miniprep, and sequencing template preparation
A105483	Ampicillin Na	PharmPure™, USP	Antibiotic for resistance screening	Used for AmpR vector transformant screening and antibiotic plate preparation
A105484	Ampicillin Na	for cell culture	Antibiotic for resistance screening	Used for AmpR vector screening, liquid culture, and clone expansion
A301887	Ampicillin Solution	100mg/ml,sterile	Antibiotic screening solution	Used for AmpR cloning plates, liquid culture, and transformant expansion
A1509554	Ampicillin Sodium Solution (50 mg/ml, Sterile)	sterile-filtered,BioReagent,for cell culture,50 mg/ml	Antibiotic screening solution	Used for molecular cloning resistance screening and rapid medium preparation
K103025	Kanamycin sulfate	≥94%(N), from Streptomyces kanamyceticus	Antibiotic for resistance screening	Used for KanR vector screening, positive clone culture, and plasmid propagation
K103024	Kanamycin sulfate	PharmPure™, USP	Antibiotic for resistance screening	Used for KanR clone screening and bacterial culture
K103026	Kanamycin sulfate	analytical standard	Antibiotic for resistance screening	Used for screening system controls and medium preparation
K432461	Kanamycin sulfate from Streptomyces kanamyceticus	BioReagent, suitable for plant cell culture, powder	Antibiotic for resistance screening	Used for KanR vector screening and transformant culture
K1509560	Kanamycin Sulfate Solution (10 mg/mL, Sterile)	sterile-filtered,BioReagent,for cell culture,sterile,10 mg/ml	Antibiotic screening solution	Used for KanR clone screening and liquid culture
K1509563	Kanamycin Sulfate Solution (50 mg/mL, Sterile)	sterile-filtered,BioReagent,50 mg/ml	Antibiotic screening solution	Used for KanR plate and liquid medium preparation
K1509565	Kanamycin Sulfate Solution (100 mg/ml, Sterile)	sterile-filtered,BioReagent,for cell culture,100 mg/ml	Antibiotic screening solution	Used for KanR clone plate and liquid medium preparation
C100331	Chloramphenicol	Moligand™, ≥98%	Antibiotic for resistance screening	Used for CmR vectors, low-copy plasmids, and compatible plasmid system screening
C100332	Chloramphenicol	Moligand™, analytical standard	Antibiotic for resistance screening	Used for CmR screening systems and antibiotic medium preparation
C100334	Chloramphenicol	Moligand™, suitable for plant cell culture	Antibiotic for resistance screening	Used for CmR vector screening and cell culture-related screening systems
C100333	Chloramphenicol	PharmPure™, USP	Antibiotic for resistance screening	Used for CmR vector transformant screening
C1509569	Chloramphenicol Solution (34 mg/mL, Sterile)	sterile-filtered,BioReagent,for cell culture,sterile,34 mg/ml	Antibiotic screening solution	Used for CmR transformant screening and low-copy vector culture
T433240	Tetracycline hydrochloride	BioReagent, for cell culture, Powder	Antibiotic for resistance screening	Used for TetR vector transformant screening and specific cloning system culture
T105494	Tetracycline hydrochloride	analytical standard	Antibiotic for resistance screening	Used for TetR screening systems and resistance-condition validation
T140622	Tetracycline hydrochloride	≥96%	Antibiotic for resistance screening	Used for TetR-related clone screening and medium preparation
T1509585	Tetracycline Hydrochloride Solution (5 mg/mL, Sterile)	sterile-filtered,BioReagent,for cell culture,sterile,5 mg/ml	Antibiotic screening solution	Used for TetR plate and liquid medium preparation
I274316	Isopropyl-β-D-thiogalactopyranoside(IPTG)	Ultra pure, ≥99%	lac inducer	Used to induce lacZα expression in blue-white screening
I104812	Isopropyl beta-D-thiogalactoside（IPTG）	≥98%	lac inducer	Used for blue-white screening and IPTG induction systems
I301904	IPTG Solution	50mg/ml，sterile	lac inducer solution	Used for blue-white screening plate preparation and IPTG induction systems
I485502	Isopropyl β-D-thiogalactopyranoside solution	ReadyMade IPTG solution for Blue-white screening	Blue-white screening reagent	Used for TA cloning and lacZα complementation screening systems
X108836	5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal)	≥98%	Blue-white colorimetric substrate	Used for lacZα complementation blue-white screening and preliminary distinction between empty vectors and candidate insert clones
X274331	X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)	Ultra pure	Blue-white colorimetric substrate	Used for blue-white screening plate preparation and positive candidate clone selection
X1520260	X-Gal Solution (20 mg/mL)	BioReagent	Blue-white colorimetric substrate solution	Used for rapid preparation of blue-white screening plates
B425669	5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside	10mM in DMSO	X-Gal solution	Used for blue-white screening and lacZα color development
E778409	Endo-free Plasmid Maxi Kit	BioReagent, for DNA and RNA applications	Plasmid extraction product	Used for endotoxin-free plasmid preparation of confirmed clones, suitable for transfection and functional experiments
G665587	GoldHi EndoFree Plasmid Maxi Kit		Plasmid maxi-prep product	Used for large-scale preparation of low-endotoxin plasmid DNA
G665655	GoldVac EndoFree Plasmid Maxi Kit		Plasmid maxi-prep product	Used for vacuum-based preparation of endotoxin-free plasmid DNA
D1088768	Endo-free Plasmid Mini Kit	BioReagent, for DNA and RNA applications	Plasmid extraction product	Used for positive clone plasmid extraction, restriction identification, sequencing, and preparation before transfection
D598099	DNA gel Recovery Kit		DNA recovery product	Used for recovering vector backbones, inserts, and target bands from restriction digestion
D1373545	DNA Purification Kit	BioReagent, for DNA and RNA applications	DNA purification product	Used for PCR product purification, digestion product purification, and pre-ligation DNA purification
U1492721	Universal DNA Purification and Recovery Kit (Spin Column)	BioReagent	DNA purification and recovery product	Used for cloning fragment purification, gel recovery, and sequencing template preparation
A1521384	DNA Gel Extraction Kit	Suitable for molecular biology, BioReagent	Gel extraction product	Used for recovering vector digestion bands, inserts, and PCR products
S1456145	UltraBio™ Small DNA Clean Beads	BioReagent, DNase, RNase free, Suitable for molecular biology, for DNA and RNA applications	Small-fragment DNA purification material	Used for short inserts, primer-dimer removal, and small-fragment purification before cloning

7 Key Considerations in Experimental Design

7.1 Distinguishing Screening Results from Final Confirmation

(1) Antibiotic-positive

Antibiotic positivity only indicates the presence of a DNA molecule carrying a replicable or expressible resistance marker. It does not prove that the target insert is correctly inserted.

(2) PCR-positive

Colony PCR positivity indicates that the amplified region matches the expected result, but it cannot exclude mutations, rearrangements, or small deletions outside the PCR-amplified region.

(3) Correct restriction digestion

A correct restriction pattern indicates that the overall structure is generally consistent with the design, but it cannot detect all point mutations or small-fragment changes.

(4) Correct sequencing

Only after sequencing covers the junctions and the complete insert can the clone be used as the main basis for downstream expression, functional experiments, or plasmid library storage.

7.2 Selecting an Appropriate Confirmation Depth

(1) Routine storage clones

If the clone is used only as an intermediate storage vector, colony PCR and sequencing of key regions may be sufficient.

(2) Expression vectors

The ORF, tag junctions, promoter-proximal sequence, and termination region should be covered by sequencing to ensure a correct reading frame.

(3) Functional mutants

The mutation site, full-length ORF, and possible PCR-introduced mutation regions must all be confirmed. Multiple independent clones should be screened if necessary.

(4) Multi-fragment constructs

Every junction should be confirmed by sequencing. A single positive PCR result for one fragment cannot be used to judge the entire construct as correct.

8 Common Questions

8.1 How many colonies should be picked for molecular cloning screening?

For routine double-digestion single-insert cloning, 6–12 colonies can be screened first. For TA cloning, blunt-end cloning, multi-fragment assembly, or large-insert cloning, the number of screened colonies should be increased. If empty-vector background is high, the vector treatment and ligation system should be optimized first rather than simply increasing the number of colonies picked.

8.2 Does colony PCR positivity mean that the clone is definitely correct?

No. Colony PCR only proves that the region covered by the primers produces the expected amplification result. It cannot exclude internal insert mutations, incorrect orientation, local deletion, or abnormalities in other vector regions. Sequencing confirmation is still required before final use.

8.3 Is sequencing still necessary if restriction digestion is correct?

Yes. Restriction digestion can determine fragment size and partial structure, but it cannot detect single-base mutations, small deletions, or point mutations within the reading frame. Plasmids used for expression, mutant construction, or functional research should be confirmed by sequencing.

8.4 Are white colonies in blue-white screening always positive clones?

No. White colonies may result from disruption of lacZα by an insert, but may also arise from vector mutations, reading-frame abnormalities, incubation effects, or substrate diffusion. White colonies are only candidate clones and should subsequently be confirmed by colony PCR and sequencing.

8.5 What should be done if sequencing shows double peaks?

Double peaks commonly arise from mixed clones, impure templates, repetitive sequences, or local secondary structures. First, isolate a single colony again by streaking, then perform plasmid miniprep and sequencing. If local double peaks remain, the sequencing primer can be changed or the region can be confirmed by sequencing from the opposite direction.

8.6 Why is there no expression even though the plasmid construct is correct?

Possible causes include promoter issues, reading frame, tag position, host strain, codon usage, protein toxicity, folding, and induction conditions. Correct construct sequence only confirms that the DNA level is accurate. Expression results still require optimization of host system, induction conditions, solubility, and functional detection.

Molecular cloning screening and identification should proceed stepwise through “candidate clone acquisition—insert confirmation—structural confirmation—sequence confirmation—functional validation.” Antibiotic resistance, blue-white screening, colony PCR, and restriction digestion can improve screening efficiency. However, for clones intended for downstream expression, functional experiments, or long-term storage, the final basis should be sequence-confirmed plasmid identity.

For more related articles, please see below:

[1] Cloning PCR experiments that do not depend on the ligation reaction

[2] TA cloning of PCR products

[3] Comparison of Chloramphenicol and Common Antibiotic Resistance Selection Systems in Molecular Cloning