Quick and Reliable TOPO-TA Kit

June 02, 2017

UC San Diego
Biomedical Sciences
Grad Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Thermo Fisher Scientific

Product Name:

TOPO TA Cloning Kit for sequencing

Catalog Number:

450030

Our lab uses this kit for sequencing potential CRISPR knockout cells. We extract genomic DNA from mammalian cells, perform PCR with Taq using the buffers provided, then ligate with the TOPO-TA cloning vector. The vector provided with the kit can be easily sequenced, which provides us the exact sequence that produced a stop codon in our gene.

Experimental Design and Results Summary

Application

TOPO-TA cloning for downstream sequencing applications.

Starting Material

Genomic DNA

Protocol Overview

Using genomic DNA, we run a PCR for our sequence of interest. PCR master mix, dNTPs, and water are supplied with the kit. The user only needs to provide the Taq polymerase, primers, and DNA. Following PCR, the actual TOPO-TA ligation process only takes 5 minutes. We add 2 uL PCR reaction product, 2 uL water (provided), 1 uL salt solution (provided) and 1 uL TOPO vector (provided) and let rest for 5 minutes at room temperature. Afterwards, we transform with TOP10 bacteria - also from Thermo.

Tips

Although you can buy competent cells with the kit, we prefer to buy separately, because they are usually used at a different frequency (in case some PCRs fail). Also, note that different Taq polymerases work differently with the PCR mix provided. We get the best results using Taq from Qiagen rather than Thermo's recommended Platinum Taq.

Results Summary

The results are very reliable. I have never had a problem with the TOPO ligation step provided with this kit. Additionally, the reaction is very quick and easy to perform.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Quick, easy, and reliable.

The Bad

Taq is not provided with the kit. Also, it would be ideal if one could by replacements for some parts of the kit (just the PCR and vector components), since we end up with way too much T3/7 ligases and control tubes.

The Bottom Line

I highly recommend this product if you are looking for a way to do high-volume sequencing from PCR product.

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