Gel Extraction Of DNA Fragments Of 20 Kb Size

April 06, 2018

University of Birmingham
Biosciences
Research Assistant

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Company:

Qiagen

Product Name:

QIAEXII Gel Extraction Kit

Catalog Number:

20021

A conventional column based Qiagen gel extraction kit was not able to purify DNA fragments of size 20 Kb from the agarose gels. I have used QIAEXII Gel extraction kit for successful extraction 20 Kb DNA fragments from the agarose gels. Recovery was 70 % of the total input with quality (260:280) of 1.87 as determined by spectrophotometer.

Experimental Design and Results Summary

Application

Purification of DNA fragments from agarose gels

Starting Material

1 microgram of DNA excised from 1 percent agarose gels

Protocol Overview

1. Excise the DNA fragments from agarose gels and weigh the gel.2. Add 3 volumes of QX1 buffer and 2 volumes of nuclease free water (if DNA fragments size you are extracting are greater than 10 Kb size).3. Then add 20 microliter of QIAEXII suspension (silica gel which binds with DNA). Mix well and place the above mix at 50 degree Celsius. Make sure the gel matrix is completely dissolved.4. Then follow exactly the Kit's instructions for washing step.5. Finally eluted DNA with 50 degree C pre-heated nuclease free water for 5 minutes at 50 degree C.

Tips

Do not vortex if your DNA fragment size to extract is more than 10 Kb during all steps of processing. Instead mix the contents by flicking the bottom of the tubes.

Results Summary

Good recovery of the 20 Kb DNA fragments from 1 percent agarose gel. Got around 620 ng from 1 microgram of DNA ran in agarose gels. Quality i.e. 260:280 ration of purified DNA was 1.87

DOI or PMID #

N/A

Additional Notes

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Summary

The Good

Simple protocol. Involves few steps of centrifugation using benchtop microfuge.

The Bad

As the fragment size of DNA increases, the recovery/yield also decreases.

The Bottom Line

Recommended kit and method for extraction of large size DNA fragments (above 10 Kb fragments and up to 30 kb) in my experience.

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