Dears,
I need help with the interpretation of why not obtain results in real-time PCR when I use Taq man SNP genotyping assay ID: rs 22756913 (IL17A) from Thermo fisher scientific
where the final volume reaction was10 microliter
Taq man SNP genotyping assay (20X) working was 0.3 microliter
DNA sample volume was 3 microliter with a concentration of 0.6 ng
the master mix was 5 microliter
nuclease-free water added to complete the total volume of 10 microliter
but when I use another SNP ID: rs 40401 from the same company (Thermo fisher ) with the same concentration and the volumes, I obtained results in real-time PCR
Regards.
Hi Noor Nihad Baqer , probable explanations are one or more of the following:
1. Your samples only contain just C or T alleles (emsembl reports A/G). Your probes are designed to detect just A or G. Different dog breed maybe?
2. DNA is degraded or contaminated with a PCR inhibitor.
3. Not enough sample, if you are using an standard master mix is better to use from 10-100 ng. Fast master mix 1-10 ng. In practice 33 ng always works well for both master mixes.
4. TaqMan assay must be 0.5- 1 uL for your 10 uL of reaction volume (at least for that particular assay)
5. Degraded probes due to light exposure or more than 3 thaw cycles.
6. Suboptimal probe design, CCC or more in a row, etc.
7. Primer dimerization due to high affinity or previous F & R mixture.
8. Change in the PCR cycle.
I hope this may be useful.
Hi Noor Nihad Baqer , probable explanations are one or more of the following:
1. Your samples only contain just C or T alleles (emsembl reports A/G). Your probes are designed to detect just A or G. Different dog breed maybe?
2. DNA is degraded or contaminated with a PCR inhibitor.
3. Not enough sample, if you are using an standard master mix is better to use from 10-100 ng. Fast master mix 1-10 ng. In practice 33 ng always works well for both master mixes.
4. TaqMan assay must be 0.5- 1 uL for your 10 uL of reaction volume (at least for that particular assay)
5. Degraded probes due to light exposure or more than 3 thaw cycles.
6. Suboptimal probe design, CCC or more in a row, etc.
7. Primer dimerization due to high affinity or previous F & R mixture.
8. Change in the PCR cycle.
I hope this may be useful.
I agree with above answer
In particular use of 2ng or less is much too little
Like the above commentator i would elect to use at least 10 x that amount for consistent robust data
If the same condition worked with another TaqMan probe, then as Ernesto Prado Montes de Oca mentioned, you may possibly have another type of mutation or your probe exposed to a source of light.
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