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Capillary electrophoresis systems are complex systems, and troubleshooting data quality can be complicated. The biggest contributors to data quality can be divided into these areas: software, chemistry, and instrument performance.

Troubleshooting software
Ensure that the run module you are using and the dye set match the chemistry and instrument setup. For example, if you are running a multicapillary instrument with a 50 cm array on it, the run module should have a “50” in the name. On a multicapillary system, BigDye Terminator v3.1 should run under Dye Set Z, BigDye Terminator v1.1 chemistry should run in Dye Set E (on the ABI PRISM 310 Genetic Analyzer, both chemistries run under Filter Set E, but require different matrices for analysis). The mobility file (DyeSet/Primer) should have the correct instrument, polymer, and chemistry in the name (e.g. KB_3130_POP7_BDTv.3.mob).

Troubleshooting chemistry and instrument performance
•Sequencing Standards: validate instrument
If Sequencing Standards fail, it suggests a possible electrophoresis problem due to array, polymer, buffer, water, septa, plastics, or maybe a hardware failure such as autosampler, laser, camera, etc.

•pGEM Control DNA: validate cycle sequencing and its clean-up
If the pGEM control reactions fail and the Sequencing Standards pass, look into potential problems with the sequencing kit, thermal cycler, and cleanup procedure.
The pGEM Control DNA and M13 (–21) primers needed to run the reactions are included with each BigDye Terminator Cycle Sequencing Kit

•Custom Internal Controls: validate PCR and its clean-up
If the Sequencing Standards and the pGEM controls passed, then look into potential problems with template quantity and quality, primers, PCR reaction and purification.

For more information on troubleshooting your data, please refer to the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: 3rd Edition, which can be downloaded from this link (https://www.thermofisher.com/us/en/home/global/forms/sanger-sequencing-guide-download-form.html).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

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No. This product is not recommended for use with low-copy number plasmids when the plasmid DNA is for use in automated fluorescent DNA sequencing. Low-copy number plasmids can be used with this system when the samples are for use in PCR amplification. However, keep in mind that the yields from a low-copy plasmid may not be readily visible on a gel.

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We would recommend using our Platinum II Taq Hot-Start DNA Polymerase, which is supplied with Platinum GC Enhancer, a special additive optimized to improve amplification of GC-rich targets (recommended for use with targets containing >65% GC). If it is important to preserve DNA sequence accuracy, we offer the high-fidelity Platinum SuperFi DNA Polymerase together with the SuperFi GC Enhancer to enhance the specificity and yield of difficult GC-rich target amplicons. AmpliTaq Gold 360 DNA Polymerase with the 360 GC Enhancer buffer has been shown to work with templates containing up to 80% GC. Alternatively, our PCRx Enhancer System can be used in conjunction with DNA polymerases, including native/recombinant Taq, Platinum Taq, and Platinum Taq High Fidelity to optimize PCR of problematic and/or GC-rich templates.

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We recommend using the Ion Torrent Ion Xpress Plus Fragment Library Kit (Cat. No. 4471269) for preparing libraries using the Ion Shear enzymatic shearing method, or the Ion Plus Fragment Library Kit (Cat. No. 4471252) if using physical fragmentation. The protocol for both library kits is included in the Ion Xpress Plus gDNA Fragment Library Preparation User Guide (Pub. No. 4471989). Please also refer to the Decision Tree for DNA Sequencing on the Ion PGM System and the Decision Tree for the Ion Proton System for more information.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

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SYBR Green PCR Master Mix (P/N 4309155) does not contain AmpErase UNG. It contains the following components: SYBR Green 1 dye, AmpliTaq Gold DNA polymerase, dNTPs (with dUTP), Passive Reference 1 and optimized buffer components; all the necessary components to perform real-time PCR in a convenient premixed 2X solution. This product has been tested on and is supported for use on an ABI PRISM Sequence Detection System. For more information, please consult the SYBR Green PCR Master Mix protocol.

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Please review the possible reasons and recommendations below:

- Incomplete digestion of pFastBac plasmid or insert DNA: Use additional restriction enzyme for digestion or purify the insert DNA.
- Incomplete or excessive phosphatase treatment of pFastBac plasmid: Optimize dephosphorylation conditions according to the manufacturer's recommendations for the phosphatase you are using.
- Poor recovery of pFastBac plasmid or insert DNA from agarose gel: Use our PureLink Quick Gel Extraction System to purify high-quality plasmid DNA from your agarose gel.
- Incomplete ligation reactions: Follow ligation conditions and optimize the reaction by varying vector:insert molar ratios (1:3, 1:1, 3:1).
- Insert contains unstable DNA sequences such as LTR sequences and inverted repeats: Grow transformed cells at a lower temperature (30 degrees C) or try a different competent cell strain such as MAX Efficiency Stbl2 Competent Cells.

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Sequencing reaction purification can be performed with BigDye XTerminator, Centri-Sep System or ethanol/EDTA precipitation. The protocols can be found in the BigDye Xterminator Purification Kit manual (https://tools.thermofisher.com/content/sfs/manuals/cms_042772.pdf), Chapter 4 of the BigDye Terminator v1.1 Cycle Sequencing Kit manual(https://tools.thermofisher.com/content/sfs/manuals/cms_041330.pdf), BigDye Terminator v3.1 Cycle Sequencing Kit manual(http://tools.thermofisher.com/content/sfs/manuals/cms_081527.pdf) or the DNA Sequencing by Capillary Electrophoresis Guide (http://tools.thermofisher.com/content/sfs/manuals/cms_041003.pdf).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

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A change in the read length is usually caused by something that either changes the migration of the DNA or the efficiency of the sequencing reaction. Some of the things that can cause this are:

• Buffer: Buffer may be prepared incorrectly or on the system for too long.

• Polymer: Polymer may have been on the system the system too long, used past the expiration date, frozen/crystallized and thawed, or mixed with water or buffer during capillary fill. There may be a mismatch between polymer type selected in software and what is installed on the system.

• Environment: Instrument may be operating outside environmental specifications in the Site Preparation Guide. You may have poor/improper instrument ventilation, or air flow may be blowing directly on the system.

• DNA quality: RNA/protein contamination may be reducing the sequencing reaction efficiency. If working with PCR product, there may be carryover of primers and/or dNTPs from the PCR reaction, or the sequencing reaction cleanup is incomplete. If using a kit that has beads in it for the purification, beads may be blocking the capillary tip. If using BigDye XTerminator Purification, heating the XTerminator reagent can cause loss of smaller products.

• DNA quantity: There may be too much DNA competing for entry into the capillary with labeled product, or excessive amounts of DNA creating a temporary blockage of the capillary.

For more information on other causes of short reads and how to address each issue, please refer to the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C). The guide can be found by searching the Thermo Fisher Scientific website with the catalog number 4305080.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

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All Applied Biosystems instrument systems are qualified to detect a two-fold change. Scientifically speaking, there is a statistical calculation that predicts the probability of detecting one molecule in any given sample called the Poisson Distribution. The Poisson Distribution predicts that one molecule will be detected 66% of the time. This is assuming that the assay has been optimized to the fullest capacity and that there are no aliquoting or pipetting errors involved. Please refer to the following paper concerning one-copy detection: Lockey, C., E. Otto, and Z. Long. "Real-time fluorescence detection of a single DNA molecule." BioTechniques 24 (1998): 744-746.

Each assay designed on one of our platforms needs to be optimized to determine the Linear Dynamic Range of the assay. An Applied Biosystems Sequence Detection System (Real-Time PCR instrument) may be able to detect one copy in a fully optimized assay. As stated above, one copy can only be detected 66% of the time in a fully optimized system.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

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Some of the causes of dye breakdown or artifacts in the data are:

• Oxidation or change in pH: Injecting out of water or old Hi-Di Formamide that has broken down. Also, some cleanup methods can oxidize the dyes.

• Array: An old or contaminated array can cause dye breakdown.

• Arcing: An arcing event on the system can destabilize the blue baseline and—in severe cases—mask it completely.

For more information on other causes of dye breakdown and artifacts in data and how to address each issue, please refer to the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C). The guide can be found by searching the Thermo Fisher Scientific website with the catalog number 4305080.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

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The Template Generation System Kit provides a method for introducing primer binding sites randomly into foreign target DNA. Insertion of the Entranceposon into foreign DNA makes it possible to sequence the flanking DNA using the primers supplied in the kit. The clones for targeted sequencing can be selected from the population of random insertions by using simple colony-PCR or restriction enzyme mapping.

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No, indexed adaptors are specifically designed for each product.

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