7 Matching Annotations
- Sep 2024
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www-nature-com.ezproxy.rice.edu www-nature-com.ezproxy.rice.edu
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we found that the clouds can become more compact and produce substantially less rain upon: (i) reducing the ramp rate to 1 °C/sec in every PCR step (Fig. 10a), (ii) increasing the annealing/extension time to 2 minutes (up from 1 minute; Fig. 10b), and (iii) increasing the number of cycles to 50 (Fig. 10c)28
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- May 2024
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www.bio-rad.com www.bio-rad.com
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Restriction Digestion
See notes in this section for restriction digestion tips
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www.bio-rad.com www.bio-rad.com
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DNA fragmentation by restriction digestion prior to dropletgeneration enables optimal accuracy by separating tandemgene copies, reducing sample viscosity, and improving templateaccessibility for input samples >66 ng per well
NEB says
Digestion is recommended whenever DNA input is greater than 75 ng Source
NEB says that biorad recommends these enzymes: AluI, CviQI, HaeIII, HindIII-HF, MseI
More guidelines
- Assemble ddPCR reactions at room temperature, this allows the restriction enzyme to digest the gDNA during the reaction set-up period
- Prepare reaction mixes as you would for a standard ddPCR reaction. Add 0.5–1 μL of each restriction enzyme (5–20 units, depending on enzyme concentration) to the reaction mixture
- After set-up, simply continue droplet generation as normal
- Restriction enzyme will be inactivated during first PCR denaturation step
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Refer to this protocol mentioned in the BioRad page, annotation
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- Feb 2023
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Digestion is recommended whenever DNA input is greater than 75 ng
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- Nov 2022
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www.biosearchtech.com www.biosearchtech.com
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For those shorter than 25 bases, a standard (dual labeled) BHQ™ Probe may be perfectly satisfactory.
Nova might be better to have for 1-step ddPCR applications where the BHQ quenchers are unstable due to reduction of the N=N azo bond by the DTT additive
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