Bst Large Fragment

Open DNA Collections Shared Reagents
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Bst Large Fragment

Reagent Sharers

Alex Brown

Summary

This enzyme comes in a pET15b plasmid. Information shared by Alex: This plasmid contains a DNA Polymerase which retains 5´ → 3´ polymerase activity from full length Bst DNA Polymerase, while lacking 5´ →3´ exonuclease activity. The polymerase has been described for use in applications requiring thermophilic strand displacement. Used for: Loop-Mediated Isothermal Amplification (LAMP), DNA sequencing through high GC regions, rapid sequencing from nanogram amounts of DNA template.

This enzyme is a codon-optimized version of Andy Ellington´s Bst LF

Expression/Production Information (if applicable)

Protocol Information

Expression and purification protocol currently available at ReClone
group in protocols.io

How to get the plasmid/material/reagent

Currently in Alex´s hands, FreeGenes and Chile/Argentina nodes
This is vector received from Alex and being used in Chile.

Terms

Under openMTA from Alex, being able to redistribute under openMTA from iBio/Chile too.

References

(PMID: 10871386, 25652028, 1793578)

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Hi everyone! Is this polymerase available for commercial use?

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Yes! It is distributed under the OpenMTA which explicitly allows commercial use. The Bst-LF US patent expired in 2016 so it is in the public domain.
Jenny

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Javier, do you have a protocol to share in protocols.io for BstLF?
(all protocols here: https://www.protocols.io/groups/recloneorg-the-reagent-collaboration-network/publications)

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I hope to have one by the end of the week. Based on Alex Brown’s protocol

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Hi, please be aware the original post :point_up_2: has been edited to include the expression and purification protocol (https://www.protocols.io/view/recombinant-protein-expression-and-purification-of-bksrkwd6).

We are currently working on a protocol for RT-LAMP based on this enzyme, MMLV and in-house prepared buffers. Preliminary results indicate that 10.7 ng/uL of BstLF (this is uL of reaction so 267.5 ng of BstLF total in a 25 uL reaction volume) is equivalent to the performance of 8U of commercial Bst2.0 (and around 125 pg/uL of MMLV, again uL of reaction so 3.1 ng of MMLV total in a 25 uL of reaction volume). We use the N2 primer set for the characterization and T7-based in vitro transcribed gene N (DNA from IDT) as a target. We used in-house prepared reaction buffers based on NEB 1X Isothermal Amplification Buffer Pack. Isaac Nuñez and Tamara Matute are now working on the optimization of these concentrations. Collabs/ideas are very welcome.

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Hi! My lab in the north of Brazil recently engaged to explore the RT-LAMP technique using commercially available enzymes and now we want to establish in house production. Would you please indicate where can I find this vector (is it available in AddGene)? Thanks

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Hi Jimmy, yes we can send you some DNA.
Could you please send us your address?
Cheers
F

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That would be really kind! May we get your email to send the informations?

Gracias/Obrigado!

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Hi Jimmy,
Ariel here, I work with Fernan and I can help you with some DNA

Write us at ariel.cerda.rojas(at)gmail(dot)com.

Regards!

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Hi All, I recently saw this new preprint where they used drying (without freezing) for an RT-LAMP reaction, and thought it might be interesting for some of you,

Not sure if this is the right place or maybe we should have a channel for Lyophilizatoin/Stability/Storage/Shipping Issues?
Happy Holidays!

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