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Should I use electrocompetent or chemically competent cells to make my library?

We recommend using electrocompetent cells for library generation, because the transformation efficiency for electrocompetent cells is higher. Higher transformation efficiency preserves library diversity.

How do I calculate transformation efficiency (TE)?

TE = (# colonies) / (µg DNA transformed) * (final transformation volume) / (volume plated) * dilution factor = cfu / µg

For example, if you transformed 1 ng of plasmid DNA into cells, diluted the transformation to a final volume of 1000 µL with Recovery Medium, then plated 25 µL of a 1:100 dilution of that transformation, and the next day you counted 150 colonies on your plate, your calculation would look like this:

# of colonies: 150
µg DNA transformed: 0.001
Final tansformation volume = 1000 µL
Volume plated = 25 µL
Dilution factor = 1 µL into 100 µL, 100-fold (factor of 100)

TE = (150 / 0.001) * (1000 / 25) * 100 = 6 x 108 cfu / µg

There are transformation protocols for chemically competent cells that do not include a heat shock. Do I need to heat shock my competent cells?

We have not tested alternative transformation protocols extensively. You can avoid the heat shock step, but it is highly likely that your transformation efficiencies will decrease.

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.

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.

Are there any limitations on the types of fluorescent reporter molecules I can use with RapiDxFire qPCR 5X Master Mix GF?

PCRAMP-012

All fluorescent dyes listed on our Fluorophore and BHQ Selection Chart are available for use with RapiDxFire qPCR 5X Master Mix GF, in which the corresponding BHQ dye has been identified.

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.

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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