Amplification Of Long Templates (15 To 20 Kb DNA Template)

November 16, 2017

University of Birmingham
School of Biosciences
Postdoctoral Research Fellow

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Company:

Sigma Aldrich

Product Name:

Expand™ Long Template PCR System

Catalog Number:

11681834001

Using Expand Long Template PCR system, 18.2 kb DNA template for construction of donor plasmid was successfully amplified. PCR was performed with 200 ng genomic DNA as template with buffer system 3 in a 50 microliter reaction. Each reaction resulted in 1.5 microgram desired template yield with good quality.

Experimental Design and Results Summary

Application

Amplification of long and difficult template for cloning

Starting Material

200 ng of Salmonella enterica Typhimurium genomic DNA per 50 microliter PCR

Protocol Overview

200 ng of genomic DNA was used in a 50 microliter PCR reaction with Buffer system 3. Performed 30 cycles of thermal cycling with 2 step amplification process mentioned in the manufacturer's instruction. Amplified PCR product was analysed by gel electrophoresis and Nanodrop.

Tips

Always thaw buffer 3 at 50 degree C for best performance of PCR reaction

Results Summary

Successfully amplified 15 and 18 kb DNA templates intended for cloning with good yield and quality as assessed by gel electrophoresis and Nanodrop measurements.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Less error prone in amplifying DNA as compared to other DNA polymerases.

The Bad

None

The Bottom Line

If you are looking for Gibson assembly cloning and need fragments of size greater than 5 kb, I strongly recommend this kit for PCR amplification long templates for cloning.

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