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Are there specific recommendations for performing 1-step RT-qPCR on RNA isolated from clinical samples?

PCRAMP-021

The nucleic acid (either DNA or RNA) will most likely need to be purified from clinical samples due to sample/transport media specific inhibitors and RNAses. For example, for RNA purification, we recommend our sbeadex‚Ñ¢ Pathogen Nucleic Acid Purification Kit for optimal purification.

Have you produced proteins at scale?

COMCEL-008

Our customers have reported growth of ClearColi strains in a 5 litre fermenter and shown optical density levels in excess of 150, and that some defoaming agents (particularly silicone-based defoamers) are beneficial while other defoaming agents such as polypropylene glycol may inhibit cell growth.

What if I don’t have a fluorimeter? How else can I monitor the reaction?

PCRAMP-009, PCRAMP-010, PCRAMP-007, PCRAMP-006

Amplified products can also be monitored by viewing the reaction on an agarose gel or by measuring the turbidity of the reaction at OD600.

Which instruments can be used to monitor the real-time fluorescent assays?

PCRAMP-009, PCRAMP-010, PCRAMP-007, PCRAMP-006

See the User Manual for a list of instruments that have been tested at Biosearch Technologies. Basically, any instrument capable of maintaining stable reaction temperatures and detecting fluorescence in the FAM (fluorescein) channel will work.

Can I use other dyes besides the Green Fluorescent Dye available in the LavaLAMP™ DNA Master Mix with Dye?

PCRAMP-009, PCRAMP-010, PCRAMP-007, PCRAMP-006

Yes. Syto-13 (Molecular Probes) and EvaGreen (Biotium) have both been tested with the LavaLAMP DNA Master Mix and they perform well.

What if I don’t have a fluorimeter? How else can I monitor the reaction?

PCRAMP-009, PCRAMP-010, PCRAMP-007, PCRAMP-006

Amplified products can also be monitored by viewing the reaction on an agarose gel or by measuring the turbidity of the reaction at OD600.

What purification scale is sbeadex Lightning suitable for?

SBXLCK, NAP40-032-00

The standard protocol assumes a 200 µL volume of lysate for each purification. The purification protocol can be upscaled or downscaled depending on laboratory requirements. We do not provide different scales of kits (e.g. mini, maxi); instead core kit reagents and additional reagents can be adjusted to suit your specific laboratory process.

Which instruments can be used to monitor the real-time fluorescent assays?

PCRAMP-009, PCRAMP-010, PCRAMP-007, PCRAMP-006

Basically, any instrument capable of maintaining stable reaction temperatures and detecting fluorescence in the FAM (fluorescein) channel will work.

Do you have any recommendations for purifying DNA from difficult biological samples such as plant leaves and seeds before shearing?

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

No. We do not have any specific recommendations for DNA purification. However, the purified DNA should be high quality with A260/280 ratios above 1.8. The DNA should also be free of detectable RNA contamination. Prior to shearing/fragmentation, the purified DNA samples can be analysed on 0.7% agarose gel to check for sample degradation and RNA contamination.

Can intercalating dyes be used with this mix?

BHQ-001

BHQ Probe Master Mix has been optimised for use with hydrolysis-probe based chemistry, for both end-point genotyping and qPCR applications. For use with intercalating dyes, we would recommend that you perform your own optimisation and validation to ensure reproducible sensitivity and specificity is achieved using your selected dye.

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