PCR and sequencing for genotyping of candidate Plasmodium falciparum artemisinin resistance SNPs in the Kelch 13 gene
D. Menard & F. Ariey Institut Pasteur
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Comments
PCR product from the first PCR reaction cannot be seen
I ran this assay and found that i cannot see PCR product from the first PCR reaction. Do ypu have any comments on this observation.
PCR product from the first PCR
PCR product from the inner PCR can be observed when you perform the PR run with a good concentration of DNA extracted from your initial sample. If the DNA concentration is too low, it is normal to not see PCR product from the inner PCR.
Primer name switching
After i did primer blasting, i found primer name switching from K-13 F to K-13 R and K13 Nested 1 F to K13 Nested 1 R vice versa.
The primer name should be corrected as followed;
- K13-F change to K13-R, K13-R change to K-13 F
- K13 Nested-F change to K13 Nested-R, K13 Nested-R change to K-13 Nested F
K13 primers names should be corrected
I do not agree that we need to change the names of the K13 primers, because the ORF of the K13 gene is oriented from 3' to 5'.
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