Hi. Everyone. I have extracted DNA from hair follicles through Brief extraction buffer and measured conc. by nanodrop (100 ng/ul) but when I used this sample for PCR, no amplification observed. After all troubleshootings I changed template (DNA was extracted through tissue by KIt) and observed amplification. At last, I run former DNA (extracted from hair follicle) on gel but no band appear. So, I can not understand why nanodrop gives concentration and no band on Gel?..Please need help in this regards.
Thanks in advance.
Hi Muhammad
Maybe you can check a few simple troubleshooting aspects to find the critical point in your proceeding.
Did you use a positiv control for PCR to exclude errors in your reaction mix?
Check the DNA concentration with a good photometric unit, perhaps your DNA is too low, even if 100ng/µl seem to fit.
is the amount of genomic DNA visible on Agaraose-gel?
Do you have a positiv control for your extraction of genomic DNA? Perhaps you have a loss there...
Do you have possibilty of microbiological pollution in your system or DNAse activity?
good luck
Hi, Frank, Thanks a lot.
In our lab, mostly use nanodrop to measure DNA conc.
When I run the these samples on gel I did not find any band.
So, I want to know what are possibilities why DNA band not appear.
Change the loading buffer. May be due to loading buffer. Once i also faced this problem.
i think , you should increase the Quantity of Sample. May be per unit level Quantity of DNA less. Thanks
Aoa, Irfan, I will try but same loading dye is working with other samples, But lets try.
Saqib Umer, Brother, I run the control with less conc. as compared to these samples but that samples amplified so it mean no issue of conc.
- Badges
- Science method