Feb 03, 2015

Public workspaceLigation Protocol for NEB PCR Cloning Kit (E1202) V.1

DOI
dx.doi.org/10.17504/protocols.io.cp5vq5
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DOI: dx.doi.org/10.17504/protocols.io.cp5vq5
External link: https://www.neb.com/protocols/2013/12/27/ligation-protocol-e1202
Protocol Citation: New England Biolabs: Ligation Protocol for NEB PCR Cloning Kit (E1202). protocols.io https://dx.doi.org/10.17504/protocols.io.cp5vq5
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: January 25, 2015
Last Modified: March 28, 2021
Protocol Integer ID: 477
Keywords: ta cloning, pcr cloning kit, topo cloning, ZeroBlunt, ligation reaction, PCR amplicon, ligase
Abstract
Ligation protocol for the PCR Cloning Kit (E1202)
Guidelines

LIGATION REACTIONPOSITIVE 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)
H2Oto 5 μl2 μl
Cloning Master Mix (2X)5 μl5 μl
Total Volume10 μl10 μl

*For purified PCR amplicon products, the amount of insert to be added can be calculated by relative length or molar calculations. Formulas below use the recommended values of 25 ng of linearized vector (2525 bp) per reaction and an insert-to-vector ratio of 3:1.

a. Relative length calculations:
ng insert to be added = (3)(25 ng vector) (bp of insert/2525 bp of vector)

b. Molar calculations:
i. 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
2525) = (25)(1000)/(650)(2525) = 25000/1641250 = 0.015 pmoles vector
ii. Calculate a 3-fold molar amount of insert to add to each ligation:
(3)(0.015 pmoles vector) = 0.045 pmoles insert
iii. 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)/1000

As 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 from reactions yielding a specific product, use 1 μl or less of the reaction as insert. Do not use larger amounts, as carryover material from the PCR reactions can inhibit ligation or transformation. To estimate the concentration of PCR product for the above calculations, compare the reaction yield to known amounts of DNA fragments in a marker lane, such as our Quick-Load® Purple 2-Log DNA Ladder (NEB #N0550S).
Materials
MATERIALS
ReagentNEB PCR Cloning Kit - 20 rxnsNew England BiolabsCatalog #E1202S
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.
Mix the first 3 components of the reaction.
Protocol
E1202 Ligation Mixture
NAME
E1202 Ligation Mixture
CREATED BY
New England Biolabs
Linearized pMiniT Vector (25 ng/μl), 1 μl
Amount1 µL
Insert, 1-4 μl
H2O to 5 μl
Add Cloning Master Mix (2X), 5 μl, to a total of 10 μl per ligation reaction.
Amount5 µL
Incubate at room temperature (25°C) for 5–15 minutes.
Note
While 5 minutes is sufficient, 15 minutes will increase transformation levels for amplicons with single base overhangs
Incubate on ice for 2 minutes.
Duration00:02:00
Transform immediately into NEB 10-beta Competent E. coli or store at -20°C.