Purification of DNA from Agarose Gels for Cloning

March 28, 2018

University of Birmingham
Biosciences
Research Assistant

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Company:

New England Biolabs

Product Name:

Monarch DNA Gel Extraction Kit

Catalog Number:

T1020S

One microgram of plasmid DNA digested with restriction enzyme was separated on 1 percent agarose gels. Expected DNA length of 4 kb plasmid backbone was purified using Monarch DNA gel extraction kit. Recovery was 70 percent with 260:280 absorbance ratio of 1.84.

Experimental Design and Results Summary

Application

Purification of DNA from agarose gels for cloning and construction of plasmids

Starting Material

1 microgram of plasmid DNA digested with restriction enzymes and separated on 1 percent agarose gel

Protocol Overview

Protocol is very simple which comes as quick reference card with the kit. Follow the kit's instructions. Dissolve the excised agarose gel containing correct sized DNA fragment with 4 volumes of gel dissolving buffer at 55 degree C. Make sure that agarose gel is been completely dissolved. Then bind the DNA into column matrix. Wash the column with wash buffer two times. Finally, elute with 10 microliter of molecular biology grade nuclease free water. I usually allow the columns with nuclease free water added for 5 minutes at room temperature so that recovery of DNA is more. Then check the quality of eluted DNA in Nanodrop.

Tips

Make sure that agarose gel is completely dissolved in dissolving buffer before loading onto column matrix to bind the DNA.

Results Summary

Using this kit, DNA was gel purified with less time on hands and quickly. Quality of the DNA was good i.e. 260:280 ratio was 1.84 though 260:230 ratio was 0.88. Gel purified DNA fragments were then used for cloning and construction of plasmids without any errors.

DOI or PMID #

N/A

Additional Notes

None

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Summary

The Good

Easy to follow protocol. Less time on hands. No need to use isopropanol and less consumption of plastics like tips/tubes.

The Bad

Only negative side, I never got good 260:230 ratio in spite of performing the extraction protocol as per kit's instruction. Kit protocol never mention about expected 260:230 ratio.

The Bottom Line

I highly recommend this kit in a molecular biology lab. Very satisfied with kit's performance. One good thing is that you can excise and gel purify DNA fragments up to 25 Kb length with at least 50- 60 percent recovery.

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