Hello,
I am running a PCR amplification using NEB Q5 high fidelity polymerase kit on 1.4-1.6 kb gBlock fragments with the following reaction conditions:
> 30s at 98°C
> 10s at 98°C - denaturation; 20s at varying temperatures (see image) - annealing; 45s at 72°C - elongation ---> repeated for 32 cycles
> 2min at 72°C - polishing
I get non-specific bands across almost all of the samples although we tried different annealing temperatures. I checked my primers for non-specific binding sites and there don't seem to be any. Do you have any advice? Could secondary structure in the template, extension time or the annealing time be an issue?
Saverio Charalambous Did you even check if your sample was contaminated? Does your laboratory perform PCR on samples from different organisms?
It is important to check this and see if there was any contamination of the samples. Also it is important to know if your matrix solution of your primers are also not contaminated.
Try using a primer that would not amplify with your samples and see if any bands come up. If it appears, it may be a sign of contamination and extensive cleaning will be required to remove contaminating amplicons.
Saverio Charalambous Could you please provide more details about your templates. Are these unknown samples or known positive controls? What does the third sample of the each lot carry. The 3rd sample seems to work fine it does not have any multiple bands.
How did you reconstitute your primers? Further, how did you conclude the specificity of your primers? Further what is working concentration of your primer? Besides what is target gene of your interest? Are you by any chance targeting the repeat sequences?
Was the DNA extracted from single source of origin (Eg. - Microbial cultures, Cell-lines, Tissue) or was it clinical specimen? In other words, could you please clarify whether there is a possibility of your eluate carrying DNA of multiple origin?
If you could provide me with the above all information then probably I can help you figuring out the problem incurred over here.
Thanks
Yugendran
Yugendran T
Goodmorning, the DNA has not been extracted from any source; it has been ordered and it is being amplified using PCR
All the samples are positive controls. Indeed no. 3 has no issue but we are most worried about no.4 and 5 (1.6 kb and 1.8 kb respectively). No 2 and 3 are exactly the same but the two fused genes are in reverse order.
The primers have been reconstituted in TE ( Tris- HCl , EDTA) and their working conentration in their final mix is 10 μM.
We used the benchling primer tool to search for non-specific annealing and from that determined there wasn’t any.
2, 3 and 5 include a protein that has repeat sequences.
Thank you for the help
Saverio Charalambous Does any of your primer flank the repeat sequences?
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