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Tech Note: Superior Amplification of Bisulfite-Treated DNA for Methylation Studie

TECH NOTE

Outperforming the TOPO Cloning Workflow in Speed and Accuracy

Data kindly provided by: Juanita Von Dwingelo
Ph.D. Candidate, University of Louisville

Overview

Juanita Von Dwingelo and her colleagues have been investigating the interactions between a protein of interest and host proteins in yeast two-hybrid studies. The TOPO cloning method has been part of their standard workflow for cloning these proteins of interest. Unfortunately, this has led to cloning experiments that take a long time and are often unreliable. When they tried using In‑Fusion Cloning to construct their plasmids, they discovered that the time it took to obtain a finished clone was reduced by two days, and had the added advantage of 100% cloning efficiency.

Researcher's Notes:

“Our lab primarily uses a lengthy and unpredictable method for cloning proteins of interest into different vectors. Previously we have used TOPO methods . . . . this is a relatively lengthy process, which from start to finish takes an average of five days. Our lab does quite a bit of cloning so it would be useful to find a quicker method to use for cloning.”

Results

A yeast two-hybrid bait vector, pGBKT7, was linearized by a double restriction digest with BamHI and SalI. The eukaryotic-like ankyrin effector, AnkH, was PCR amplified from L. pneumophila with primers that incorporated cloning ends specific to the linearized vector. The PCR amplicon was gel purified and then quantified for use in the In-Fusion cloning reaction with the linearized vector. The competent cells included in the kit were transformed with the cloning reaction, and the resulting colonies were screened via PCR of miniprep DNA isolated from overnight cultures. The total time for the cloning protocol was three days, and ten out of ten colonies tested were shown to contain the correct insert.

Researcher's Notes:

“I would definitely use [In-Fusion Cloning] again as it was a very simple and quick process compared to other cloning methods that take more time and aren’t as reliable.”

Conclusions

In-Fusion technology improved the cloning workflow in terms of speed and accuracy for cloning proteins of interest for yeast two-hybrid experiments. The observed cloning efficiency was 100%, in contrast to a previous TOPO cloning method that yielded only 50–75% efficiency at best. Additionally, it took less time to perform cloning using the In-Fusion system, which produced the desired construct in just three days instead of five.

Methods

An E. coli culture containing the pGBKT7 vector was grown at 37°C with shaking overnight in the presence of ampicillin. Plasmid DNA was isolated using the Wizard Plus SV Minipreps DNA Purification System (Promega) and then quantified. The vector was linearized via restriction digest with BamHI and SalI for one hr at 37°C, according to the manufacturer’s instructions.

The fragment of interest (AnkH) was amplified from Legionella pneumophila strain AA100 with primers containing cloning ends compatible with the linear vector and necessary for In‑Fusion cloning. Primers were designed using Clontech’s online primer design tool. The PCR reaction was set up as shown below.

12.5 µl CloneAmp HiFi PCR Premix
1.5 µl Forward Primer
1.5 µl Reverse Primer
5 µl DNA template
4.5 µl Nuclease-free water
25 µl Total Volume

PCR was performed with denaturation at 98°C x 10 sec, annealing at 55°C x 15 sec, and extension at 72°C x 1 min, for a total of 35 cycles.

The PCR product was run on a 1% agarose gel, purified with the provided Macherey-Nagel Nucleospin Gel and PCR Clean-Up kit, and then quantified. In-Fusion cloning reactions, including positive and negative controls, were set up as recommended in the user manual (see table below). The insert and linearized vector for the positive control reaction were both provided with the In‑Fusion HD Cloning Plus system.

In-Fusion Cloning Reaction Setup
Component AnkH Experiment Positive Control Negative Control
5X In-Fusion HD Cloning
Plus Enzyme Premix
4 µl 2 µl 2 µl
Linearized vector 4 µl
(pGBKT7 +
BamHI + SalI)
2 µl
(pUC19 Control
Vector)
4 µl
(pGBKT7 +
BamHI + SalI)
Insert 5 µl
(AnkH PCR product)
1 µl
(Control Insert)
0 µl
Water 7 µl 5 µl 4 µl
Total volume 20 µl 10 µl 10 µl

In-Fusion cloning reactions were incubated for 15 min at 50°C. The provided Stellar Competent Cells were transformed with the reaction mixtures. Transformations were plated on LB plates containing 100 µg/mg ampicillin and incubated overnight at 37°C. Colonies were picked for overnight cultures in LB + ampicillin media and grown overnight at 37°C with shaking at 250 rpm.

Plasmid DNA was isolated from overnight cultures using the Wizard Plus SV Minipreps DNA Purification System. PCR was then used to determine the presence of the insert in each clone. Both positive and negative experiments produced the expected results. Ten out of ten experimental colonies were confirmed to contain the AnkH fragment.

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