Gel Purification of PCR Amplified DNA

July 19, 2018

University of Birmingham
School of Biosciences
Postdoctoral Research Fellow

Overall

Quality of Results

Ease-of-Optimization

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Company:

New England Biolabs

Product Name:

Monarch DNA Gel Extraction Kit

Catalog Number:

T1020S

I have used Monarch DNA gel extraction kit for purification of the PCR product of size 15 Kb. Extraction went well and good recovery of 15 Kb product with 70 % recovery after Gel extraction. Quality was good with Nanodrop 260:280 ratio close to 1.85.

Experimental Design and Results Summary

Application

DNA extraction from 0.8% agarose gel run

Starting Material

150 mg of 0.8 % agarose gel excised for 15 Kb DNA band per gel extraction column

Protocol Overview

The protocol is very simple and easy. I followed exactly the protocol which come with kit. Once gel dissolving buffer has been added to excised gel, dissolve the gel completely for 10 minutes at 50 degree Celsius for 10 minutes. Then put dissolved gel and buffer mix into purification columns and follow the remaining steps mentioned in the protocol. Finally elute DNA with 20 microliter of nuclease free water. After elution, check quality of the DNA by Nanodrop spectrophotometer.

Tips

Dissolving the gel completely is critical step for good recovery and without gel contamination.

Results Summary

After using Monarch DNA gel extraction kit, I was able to successfully gel purify PCR products of size 15 Kb in a column based purification protocol. This was not achieved in Qiagen PCR purification columns (which is having limitation to extract/purify only up to 10 Kb PCR product). Recovery was nearly 70% with good quality as checked by Nanodrop spectrophotometer measurement of 260:280 ratio.

DOI or PMID #

N/A

Additional Notes

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Summary

The Good

Very quick and easy to follow protocol. Can be easily extract/gel purify the DNA product up to 25 Kb size.

The Bad

Sometimes recovery is less than expected and 260:230 ratio was poor after extraction.

The Bottom Line

Overall, it serves my main purpose of extracting and gel purifying DNA fragments of size greater than 15 Kb up to 20 Kb in column based purification method (which is not possible in Qiagen column based method). I recommend this kit if want quick and easy extraction of larger DNA fragments.

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