The liquid enzyme should be stored at -20°C for at least 12 months without activity loss.
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Problem
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Probable Cause
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Solution
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Few or no colonies are obtained from the transformation.
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The competent cells have low transformation efficiency.
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Check the transformation efficiency. Competent cells with >1×108 cfu/μg are recommended.
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Too much reaction mixture is used.
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Do not add more than 20 μl of reaction mixture to 50 μl of competent cells. Too much reaction mixture inhibits the transformation.
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There are inhibitory contaminants from PCR DNA or from linearized vector. The molar ratio of vector to insert is off.
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Both the PCR DNA and the linearized vector should be purified. Usually an insert/vector molar ratio of 2:1 is optimal. If the insert is as large as the linearized vector, a molar ratio of 1:1 can also be used.
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Too long or short a recombination time.
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It is recommended to keep the recombination procedure within 30 min.
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The linearized cloning vector or primer is large.
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Connect the product to the target step recombinant vector.
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The cloning vector is not completely linearized.
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Gel-purify the linearized vector.
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The cloning reaction is contaminated with plasmids having the same antibiotic resistance.
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Purified PCR DNA may contain the template plasmid, so gel-purify the PCR DNA.
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Most of the colonies contain no insert.
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The cloning vector is not completely linearized.
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Gel-purify the linearized vector.
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The cloning reaction is contaminated with plasmids having the same antibiotic resistance.
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Purified PCR DNA may contain the template plasmid, so gel-purify the PCR DNA.
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