Hi
I am working on a project where I have specific segments of interest. Almost 18 region of TB positive samples were selected from where the mutation can be occurred. For that mutation the specific drug could be resistant. So i wanted to sequenced that specific region applying mini seq illumina sequencing. So i extracted the DNA from left over through Qiagen extraction kit. But when I go for quantification through qubit fluorometer i was surprised that the quantity of the sample is very low . I am confused why this happens. Even though when i pcr that product the quantity is also low . But my pcr was good because the positive control result is perfect. So anyone know or have better solution on that ,please kindly help me
Thanks
If I am understanding this properly, it seems your sample input is too low. Can you obtain more cells and prepare more genomic DNA?
If you cannot obtain more DNA, then try adjusting your primer concentrations. Due to low DNA input, your primer concentrations could be too high for this PCR. I also recommend running the DNA products out on a TBE gel. Check to see the amount of primers remaining relative to the PCR product. Also, you probably need to adjust the number of cycles.
Thank you so much .But we are using pcr kit. All the necessary thinks are mixed up together . So its not possible to check the primer concentration. I think changing the cycle can be a good option although we are maintaining the standard protocol .
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