Setting Up PCR Reactions Using i7 and i5 Indexing Primers

October 04, 2017

University of Birmingham
School of Biosciences
Postdoctoral Research Fellow

Overall

Performance

Ease-of-Optimization

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Company:

Illumina

Product Name:

TruSeq Index Plate Fixture

Catalog Number:

FC-130-1005

During high throughput whole genome sequencing library preparation using NEBNext Ultra II DNA library prep kit, incorporation of i7 and i5 indexing primers to each sample in the well of 96 microplate requires considerable patience and skill without messing up with samples and primer combination. Use of TruSeq Indexing plate fixture is highly advantageous as there will be confusions in addition of primers in the sample wells and we can keep track of samples with appropriate primers being added in an organised way this fixture.

Experimental Design and Results Summary

Application

Setting up PCR reactions using i7 and i5 primers

Starting Material

NEBNext Ulta II adapter ligated DNA libraries

Protocol Overview

Arrange (orange cap) i7 primers in increasing order horizontally, sothat the lowest number i7 index primer is in column 1, second lowestnumber i7 index primer is in column 2. Arrange (white cap) i5 primers in increasing order vertically, so thatthe lowest number i5 index primer is in row A, second lowest numberi5 index primer is in row B.Record their positions on the PCR setup template.Using a multichannel pipette, add desired volume of i5 primers to every column (as needed) of the PCR plate.Discard the original i5 white caps and apply new caps to avoid indexcross-contamination.Using a multichannel pipette, add desired volume of i7primers to every row (as needed) of the PCR plate. .Discard the original i7 orange caps and apply new caps to avoid indexcross-contamination. Add NEBNext PCR Master Mix to each well that containsprimers and proceed for required thermal cycling programme.

Tips

It is critical to change tips between columns and as well in rows to avoid cross-contamination.

Results Summary

Multiple samples in 96 well microplate were amplified using PCR without any confusion or messing up with addition of primers to samples.

Features Summary

Avoids wrong incorporation of indexing primers to the samples especially during high throughput sample prep.

DOI or PMID #

N/A

Additional Notes

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Summary

The Good

Ideal and handy tool for setting up PCR reactions using Illumina i7 and i5 indexing primers during sequencing library preparation.

The Bad

Nothing

The Bottom Line

Highly recommended to use TruSeq indexing plate fixture to set up PCR reactions during genome sequencing library preparation.

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