Ligation Protocol for NEB PCR Cloning Kit (E1202)
New England Biolabs
Published: 2022-02-23 DOI: 10.17504/protocols.io.be6cjhaw
Abstract
Ligation protocol for the PCR Cloning Kit (E1202).
Before start
For purified PCR amplicon products, the amount of insert to be added can be calculated by relative length or molar calculations. See the Guidelines for the formulas.
Steps
1. This ensures the ligase is not allowed to recircularize the vector backbone before this insert is present. It is recommended that first-time users of this kit perform the positive control ligation reaction.
*For purified PCR amplicon products, the amount of insert to be added can be calculated by relative length or molar calculations. For illustrative purposes calculations are shown below; however, the *For purified PCR amplicon products, the amount of insert to be added can be calculated by relative length or molar calculations. For illustrative purposes calculations are shown below; however, the NEBiocalculator web tool is a quick and convenient way to determine the insert amounts for all cloning reactions. Formulas below use the recommended values of 25 ng of linearized vector (2588 bp) per reaction and an insert-to-vector ratio of 3:1. is a quick and convenient way to determine the insert amounts for all cloning reactions. Formulas below use the recommended values of 25 ng of linearized vector (2588 bp) per reaction and an insert-to-vector ratio of 3:1.Relative length calculations: ng insert to be added = (3)(25 ng vector) (bp of insert/2588 bp of vector)Molar calculations: Convert the 25 ng vector present in the ligation reaction to pmoles: (25 ng vector)(1000)/(650 daltons per base pair)(number of base pairs in vector or 2588) = (25)(1000)/(650)(2588) = 0.015 pmoles vector Calculate a 3-fold molar amount of insert to add to each ligation: (3)(0.015 pmoles vector) = 0.045 pmoles insert Convert the pmoles insert amount to ng insert to be added: ng insert to be added = (0.045 pmoles insert)(base pairs in insert)(650 daltons per base pair)/1000As examples, these calculations will yield insert levels of 15 ng (500 bp insert), 30 ng (1 kb insert) or 60 ng (2 kb insert).For unpurified PCR amplicons, analyze 5% of your reaction by agarose gel electrophoresis both to confirm the specificity of the product and to estimate the DNA concentration of the product by comparing amplicon yield to known amounts of DNA fragments in a marker lane, such as our Quick-Load® Purple 1 kb Plus DNA Ladder (For unpurified PCR amplicons, analyze 5% of your reaction by agarose gel electrophoresis both to confirm the specificity of the product and to estimate the DNA concentration of the product by comparing amplicon yield to known amounts of DNA fragments in a marker lane, such as our Quick-Load® Purple 1 kb Plus DNA Ladder (NEB #N0550). This quantitation allows estimating the appropriate amount of PCR volume to achieve a 3:1 molar ratio of insert:vector backbone. Both too low a level of inset, or such high level of insert that insert ligates to both ends of the linearized vector, will decrease cloning efficiency. Do not use more than 1 μl of a PCR for cloning reactions to avoid carrying over PCR components that will interfere with cloning.). This quantitation allows estimating the appropriate amount of PCR volume to achieve a 3:1 molar ratio of insert:vector backbone. Both too low a level of inset, or such high level of insert that insert ligates to both ends of the linearized vector, will decrease cloning efficiency. Do not use more than 1 μl of a PCR for cloning reactions to avoid carrying over PCR components that will interfere with cloning.
Assemble ligation reactions using the chart below as a guide. Mix the first 4 components before adding 5µL consisting of 4µL and 1µL, for a total of 10µL per ligation reaction.
Note
| A | B | C |
|---|---|---|
| LIGATION REACTION | POSITIVE CONTROL | |
| Linearized pMiniT Vector (25 ng/μl) | 1 μl (25 ng) | 1 μl (25 ng) |
| Insert* | 1–4 μl* | – |
| Amplicon Cloning Control (1 kb) (15 ng/μl) | – | 2 μl (30 ng) |
| H2O | to 5 μl | 2 μl |
| Cloning Master Mix (2X) | 5 μl | 5 μl |
| Total Volume | 10 μl | 10 μl |
Note
2. While 5 minutes is recomended, 15 minutes will increase transformation levels for inserts suspected as being difficult to clone.
Incubate at Room temperature (25°C) for 5-15 minutes.
Note
3.
Incubate On ice for 0h 2m 0s.
