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Agilent SurePrint Oligo Library Synthesis Amplification Guidelines PDF

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Summary of Content for Agilent SurePrint Oligo Library Synthesis Amplification Guidelines PDF

Agilent SurePrint Oligo Library Synthesis (OLS) Amplification Guidelines For Research Use Only. Not for use in diagnostic procedures.

Please note that OLS is a research reagent. Agilent does not provide specific protocols for the product.

Materials and storage

Library yield 10 pmol of linear, unamplified, pooled oligonucleotides

Resuspension buffer TE, pH 8

Oligonucleotide Library storage Store the Oligonucleotide Library at 20C. The oligonucleotides (lyophilized or resuspended) are stable for at least 24 months when stored at 20C. Long term storage at 80C is also appropriate. Agilent recommends resuspending the oligonucleotides prior to storage. If you anticipate needing to repeatedly amplify the Oligonucleotide Library, aliquot the library into working volumes prior to storage to avoid multiple freeze/thaw cycles.

Amplification guidelines

In order to create a plasmid library, you must first PCR-amplify the Oligonucleotide Library, and then clone the amplicons into a suitable linearized plasmid.

Optimization of the PCR protocol to amplify the Oligonucleotide Library is needed to ensure high-quality amplicons. When optimizing the PCR conditions, consider the following guidelines. Agilent recommends using higher amounts of OLS template (200 pM) while minimizing the

total number of amplification cycles (<15 cycles). These conditions help ensure adequate representation of all the sequences in the library.

Over-amplification can lead to high molecular weight artifacts. If you observe this issue, try adjusting the number of cycles in the PCR cycling protocol and/or the concentration of the OLS library in the PCR reaction.

Keep the final volume of the PCR reaction at 50 l or less, and increase the number of replicates in order to increase yield. One reaction should be adequate to prepare multiple plasmid libraries.

Use high-quality PCR primers with lengths appropriate for the downstream application. Use a high-fidelity polymerase that also provides a high yield of amplicon, such as Agilents

Herculase II Fusion DNA Polymerase and accompanying 5 Herculase II Reaction Buffer (Agilent p/n 600675, 600677, 600679). Do not include DMSO in the reactions.

Once amplified, you need to purify the library before using it in the cloning reaction. SPRI magnetic bead purification, or comparable bead purification method, is recommended for amplicons longer than 100 bp. A final concentration of 5 ng/l or higher is required for optimal cloning efficiency, so this step may also be used to concentrate the sample as well as purify it.

The tables below offer suggested starting points for PCR optimization of reactions that use Herculase II Fusion DNA Polymerase. A well optimized reaction yields a robust band of the correct size on an Agilent BioAnalyzer DNA 1000 chip or comparable electrophoresis-based analysis method.

Table 1 Suggested reagent concentrations for PCR with Herculase II Fusion DNA Polymerase

Reagent Final concentration in the PCR reaction

OLS library 200 pM

dNTP mix 1 mM (250 M of each NTP)

Forward primer 250 nM

Reverse primer 250 nM

Table 2 Suggested cycling conditions for PCR with Herculase II Fusion DNA Polymerase

Number of cycles Temperature Duration

1 95C 2 minutes

15 95C 20 seconds

55C 20 seconds

72C 30 seconds

1 72C 3 minutes

2 Agilent OLS Amplification Guidelines

Revision A0, February 2022

*G7263-90000* G7263-90000

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Agilent Technologies, Inc. 2022

No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) wit

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