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My AmpliScribe reaction had excellent yields, but when I ran it on a native gel the RNA was much shorter than the original template. What could have gone wrong?

AS3107, MBTOOL-007

Single stranded in vitro transcripts are best separated using denaturing gels1. Denaturing gels allow in vitro transcripts to separate on the basis of their length rather than based on their length + secondary structure. When using native gels, sample migration may be altered by secondary structures in the transcripts.

What is the best way to quantify DNA isolated using the QuickExtract Plant DNA Extraction Solution?

QEP70750

Because there is residual degraded RNA in the sample, using spectrophotometric methods (e.g., A260) to quantify the DNA will give an artificially high estimate of the DNA concentration. The best method to quantify the DNA is by fluorimetry using a DNA-specific dye, such as Hoechst 33258 (bisbenzimide), or PicoGreen® dye (Thermo Fisher Scientific). These dyes bind specifically to double-stranded DNA and not to nucleotides, single-stranded DNA, or RNA.

My QuickExtract Solution was left on the bench overnight, will it be OK?

QEP70750

The QuickExtract Plant DNA Extraction Solution will still perform acceptably if left at ambient temperature for several hours. Recommended storage is at -20°C in a freezer without a defrost cycle. For convenience of use aliquots may be stored at +4°C for up to one month.

Index hopping and misassignment of reads to the wrong sample is an issue for me. Since the NxSeq HT Dual Indexing Kit creates libraries that share the same indexes on one end or the other, is there a way that I can use these Dual Indexing Primers to create libraries with truly unique indexes at both ends, with no shared indexes across libraries?

NXGSEQ-003, NXGSEQ-004, 15300-1, NXGSEQ-001, NXGSEQ-002

Yes. Since our Dual Index Primers are provided in individual tubes, you can make and multiplex up to 8 libraries with unique dual indexes at each end. There are various combinations that will produce uniquely dual indexed libraries. For example, here are (8) unique primer combinations: (701,501), (702,502), (703,503), (704,504), (705,505), (706,506), (707,507), and (708,508). In this case, you can make and multiplex up to 8 libraries with unique dual indexes at each end. The 500 Series can also be combined with the other 700 Series Primers to more fully utilize the available primers. For example, here are two sets of four libraries that can be constructed and pooled with unique dual indexes. Set 1: (709,501), (710,502), (711,503), and (712,504). Set 2: (709,505), (710,506), (711,507), and (712,508). By using these three sets of primer combinations on separate sequencing runs, you can use all of each primer provided in the NxSeq HT Dual Indexing Kit.

Which FailSafe PreMix should I use for my GC-rich template? I don't want to test all of the PreMixes.

PCRAMP-020, FS99060, PCRAMP-018, PCRAMP-017

It is nearly impossible to determine, without experimentation, which FailSafe PCR PreMix will work best with a given template/primer combination. We strongly recommend using all of the PreMixes for optimisation. Without testing the complete set of FailSafe Premixes, you may not be able to determine the optimal PCR conditions for the specific amplicon that you need.

I want to amplify a template that is ~20 kb. Should I use the FailSafe PCR System or the MasterAmp™ Extra-Long PCR Kit?

FS99100, FS99250, FS9901K, PCRAMP-018

The FailSafe PCR System can amplify templates ~20 kb. For templates >10 kb, we recommend using 2.5 units of the FailSafe PCR Enzyme Mix (for templates <10 kb, use 1.25 units) in 50 µL reactions.

What is the best way to quantify DNA isolated using the QuickExtract DNA Extraction Solution?

B009, QEF81050

Because there is residual degraded RNA in the sample, using spectrophotometric methods (e.g., A260) to quantify the DNA will give an artificially high estimate of the DNA concentration. The best method to quantify the DNA is by fluorimetry using a DNA-specific dye, such as Hoechst 332581 (bisbenzimide), or PicoGreen® dye (Thermo Fisher Scientific). These dyes bind specifically to double-stranded DNA and not to nucleotides, single-stranded DNA, or RNA.

What is the best way to quantify DNA purified using the MasterPure Gram Positive Kit?

MGP04100, MB711400

Spectrophotometric methods (e.g., A260) to quantify DNA or RNA, although in common use, may overestimate the concentration. The best method to quantify DNA is by fluorometry using a DNA-specific dye, such as Hoechst 33258 (bisbenzimide), or PicoGreen® dye (Thermo Fisher Scientific). These dyes bind specifically to double-stranded DNA and not to nucleotides, single-stranded DNA, or RNA.

What is the best way to quantify DNA or RNA isolated using the MasterPure Yeast Kits?

MPY03100, MPY80200

Spectrophotometric methods (e.g., A260) to quantify DNA or RNA, although in common use, may overestimate the concentration. The best method to quantify DNA is by fluorometry using a DNA-specific dye, such as Hoechst 33258 (bisbenzimide), or PicoGreen® dye (Thermo Fisher Scientific). These dyes bind specifically to double-stranded DNA and not to nucleotides, single-stranded DNA, or RNA. To quantify RNA, we recommend using a Qubit™ fluorometer (Thermo Fisher Scientific) or a 2100 BioAnalyzer® instrument (Agilent).

What is the best way to quantify DNA or RNA isolated using the MasterPure Complete Kit?

MC89010, MC85200

Spectrophotometric methods (e.g., A260) to quantify DNA or RNA, although in common use, may overestimate the concentration. The best method to quantify DNA is by fluorometry using a DNA-specific dye, such as Hoechst 332581 (bisbenzimide), or PicoGreen® dye (Thermo Fisher Scientific). These dyes bind specifically to double-stranded DNA and not to nucleotides, single-stranded DNA, or RNA. To quantify RNA, we recommend using a Qubit™ fluorometer (Thermo Fisher Scientific) or a 2100 BioAnalyzer® instrument (Agilent).