Site-directed mutagenesis (SDM) can be challenging, particularly during detection/confirmation of (SDM) in colonies by sequencing or PCR techniques. This common issue in SDM is heavily relying on designing of mutagenic primer pairs. The best solution is to design the mutagenic primers that have extended 3'-ends/3'-overhang. This would provide the annealing region between the mutagenic primer pair is essentially shorter. and hence ensure a lower annealing temperature for the primer pair along with a higher chance of annealing to the template.
Site-directed mutagenesis (SDM) can be challenging, particularly during detection/confirmation of (SDM) in colonies by sequencing or PCR techniques. This common issue in SDM is heavily relying on designing of mutagenic primer pairs. The best solution is to design the mutagenic primers that have extended 3'-ends/3'-overhang. This would provide the annealing region between the mutagenic primer pair is essentially shorter. and hence ensure a lower annealing temperature for the primer pair along with a higher chance of annealing to the template.
A PCR reaction consists of the template DNA, two primers covering the amplification site, an enzyme, and buffers. Multiplexing such a reaction amplifies the design challenges where one target requires 3 primers, which should be exclusively bound nowhere in the template DNA or to each other. Similarly, two targets require 6, three require 9, and so on. Each amplicon needs to be either a different size (for gels) or labeled with a different fluorescent tag that is spectrally distinct from the others in the reaction. Further complicating this, different targets in the reaction can compete with each other for resources and causes more challenges in the detection of amplicons. However, with proper primer designing, their validation, optimize quality and concentration of the enzyme and buffers certainly lead to a successful multiplex PCR reaction.
Get tips on using APO-BrdU™ TUNEL Assay Kit, with Alexa Fluor™ 488 Anti-BrdU to perform DNA Damage Assay HeLa
Get tips on using APO-BrdU™ TUNEL Assay Kit, with Alexa Fluor™ 488 Anti-BrdU to perform DNA Damage Assay U-2 OS
Site-directed mutagenesis (SDM) can be challenging, particularly during detection/confirmation of (SDM) in colonies by sequencing or PCR techniques. This common issue in SDM is heavily relying on designing of mutagenic primer pairs. The best solution is to design the mutagenic primers that have extended 3'-ends/3'-overhang. This would provide the annealing region between the mutagenic primer pair is essentially shorter. and hence ensure a lower annealing temperature for the primer pair along with a higher chance of annealing to the template.
I have extracted RNA from brain tissue but my RNA concentrations are as low as 5ng/ml with my highest being around 80ng/ml. Will I be able to perform cDNA conversion with concentrations as low as these?
Isolating DNA from tissues and paraffin-embedded tissue samples can be challenging as double-stranded DNA is physically fragile and highly susceptible to exo- and endonucleases. The best solution is to slice the tissues into smaller pieces and make a homogenate solution (using tissue homogenizer or grinding liquid nitrogen frozen samples) in the presence of DNAse inhibitors. Further, extracting DNA from the nucleus need specific methods by combining physical, mechanical and chemical lysis approaches,
Isolating DNA from tissues and paraffin-embedded tissue samples can be challenging as double-stranded DNA is physically fragile and highly susceptible to exo- and endonucleases. The best solution is to slice the tissues into smaller pieces and make a homogenate solution (using tissue homogenizer or grinding liquid nitrogen frozen samples) in the presence of DNAse inhibitors. Further, extracting DNA from the nucleus need specific methods by combining physical, mechanical and chemical lysis approaches,
Isolating DNA from tissues and paraffin-embedded tissue samples can be challenging as double-stranded DNA is physically fragile and highly susceptible to exo- and endonucleases. The best solution is to slice the tissues into smaller pieces and make a homogenate solution (using tissue homogenizer or grinding liquid nitrogen frozen samples) in the presence of DNAse inhibitors. Further, extracting DNA from the nucleus need specific methods by combining physical, mechanical and chemical lysis approaches,
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