Purification of PCR DNA Samples for Sequencing

June 15, 2020

University of Birmingham
School of Biosciences
Postdoctoral Research Fellow

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Quality of Results

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Company:

New England Biolabs

Product Name:

Monarch DNA & PCR Clean Up Kit

Catalog Number:

T1030S

Several reaction volumes of PCRs were pooled. After pooling, it was ideal to purify it from unused dNTPs, salts, and polymerase enzyme from the reaction before proceeding to down stream application such as ligations and sequencing. I used Monarch DNA and PCR Clean up kit and was able to purify my PCR reactions. Up to 5 microgram of the DNA was used per spin column and got recovery of 80% for DNA size around 5 kb.

Experimental Design and Results Summary

Application

Purification of PCR DNA for sequencing and ligation reaction

Starting Material

Pooled PCR reactions of around 5 microgram per DNA clean up spin column

Tips

None

Results Summary

DNA fragment size of 5 kb were used for purification after PCR. Used 5 microgram of total DNA per DNA clean up spin column and followed rest of the protocol according to manufacturer's instructions. Eluted finally in reaction volume of 10 microliter using molecular biology grade nuclease free water. Eluted DNA had 80% recovery with quality i.e. 260:280 was 1.8. However the 260:230 was always around 1 to 1.2 in-spite of all care and protocols followed according to instructions.

DOI or PMID #

N/A

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Summary

The Good

Quick and easy to perform kit. Less time on hands. Elution volume can be as low as 6 microliter to get concentrated DNA.

The Bad

Only negative side is that the 260:230 (quality parameter for salt/buffer carry over) was never achievable to 1.8 as mentioned in the kit.

The Bottom Line

It is a good kit but not as good as other leading providers such Qiagen.

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