CloneEZ^® PCR Cloning Kit

A: CloneEZ^® PCR Cloning Kit can cut down PCR Cloning procedure to 30 min from traditional 3.5 hrs, with one-step procedure instead of multiple steps.

	CloneEZ® Seamless Technology			Classical Cloning
Directional	√			Restriction digestion Gel extraction Ligation
Restriction enzymes not required	√			X
Ligase not required	√			X
Reaction Time	0.5 hr			>3.5 hr
Reagents Included	CloneEZ® enzyme (50 μl, 5 U/μl) 10X Buffer (100 μl) pUC57 Linear Vector (Kpnl/HindIII, 10 μl, 100 ng/μl) 1 kb contract insert ( 10 μl, 100 ng/μl)			Ligase enzyme 10X Buffer
High-efficiency cloning with long fragments (15-20 bp recommend)	4 kb	2 kb	1 kb	NA
	93%	95%	100%

B: Shown below is number and percentage of positive clones obtained with the CloneEZ^® Kit.

	1 kb	2 kb	4 kb
Number of positive clones	~1200	~800	221
Recombinant rate (%)	100%	95%	93%

C: Shown below is PCR screening result of positive clones obtained with the CloneEZ^® Kit.

General Questions

• What are the differences between the CloneEZ^® kit and the PCR Cloning Kit?
  A: CloneEZ^® Seamless cloning technology offers high efficiency, quick and direct cloning, bypassing all the tedious procedures of traditional cloning that often involve restriction, ligation, and sometimes phosphorylation. This technology works with any DNA sequence and any vector. The only requirement is that your PCR primers have to have a sequence containing 15 or more nucleotides homologous to vector sequence.
  
  
• The technical information in patent document is not available in the manual, why is that?
  A: Currently, the patent on CloneEZ^® method is invalid, so we can't provide more details about the patent technical information to the general public, and we will not show more detailed explanation in the manual until the patent is valid.
  
  

Transformation

• Why don't I get sufficient colonies when the CloneEZ^® reaction is transformed?
  A: This may be caused by four factors.
  1) The competent cells have low transformation efficiency.
  2) Too much reaction mixture is used.
  3) Presence of Inhibitory contaminants from PCR DNA or linearized vector.
  4) The molar ratio of vector to insert is off.
  Please refer to the manual for TROUBLESHOOTING.
  
  
• Why do I get incorrect colonies when the CloneEZ^® reaction is transformed?
  A: Two factors may be involved.
  1) The cloning vector is not completely linearized.
  2) The cloning reaction is contaminated with plasmids having the same antibiotic resistance.
  
  

Primer Design

• What factors need to be considered when designing the primer?
  A: Two sets of primers are used to amplify the gene of interest:
  1) 15-bp homology regions to the vector, flanking the cloning site into the insert.
  2) Specific gene sequence. The start of the 15 bp homology must begin from the 5' most extension to include the overhang, if the sequence has 5' overhang; if the sequence has a 3' overhang, homology should begin where the DNA becomes double-stranded.
  Please refer to the manual for illustration of primer design. low as 20%-30%, and it will lead to a direct repeat deletion.
  
  
• To be compatible for CloneEZ^® cloning method, what should the purity of my primer?
  A: Desalted oligos (from a qualified supplier) are very suitable for cloning with CloneEZ^® PCR cloning Kit.
  
  
• Can multiple fragments be cloned into a single vector using this kit?
  A: Yes, CloneEZ^® PCR Cloning Kit can be used for multiple fragments recombination, if there are no many repeated sequences among multiple fragments. But we still recommend to recombinate one by one after several performances of recombining 2 and 3 fragments, the efficient is low as 20%-30%, and it will lead to a direct repeat deletion.
  
  
• Can I use CloneEZ^® PCR Cloning Kit with my own vectors?
  A:Yes. GenScript's CloneEZ^® PCR Cloning Kit has been tested with any commercial or non-commercial vectors several times, and you will have no problem with it.
  
  
• What factors need to be considered when using CloneEZ^® PCR Cloning Kit with customers' vector?
  A: We recommend linearizating your vectors before using CloneEZ^® PCR Cloning Kit. Once linearized, the columned and gel-purified vector is ready for CloneEZ^® reaction.
  
  
• Will the CloneEZ^® Cloning reaction work more efficiently if I use primers that contain a longer than 15 base region of homology?
  A: 15-20 bp of homology is recommended.
  
  
• How is the stability of the enzyme included in CloneEZ^® PCR Cloning Kit?
  A: The liquid enzyme should be stored at -20°C for at least 12 months without activity loss.
  
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