I'm working on a project involving a new protocol for skeletal DNA extraction (usually poor quality DNA in the first place..) and I am using agarose gels to visualize amplifications of a 100bp region we use in our lab to assess amplification success. Unfortunately, I'm getting some results that I've never seen with my gels before, and was wondering if I could get some interpretation help.
I'm using 1.7% agarose gels and 1x TBE buffer solution. Gels are run for about 25-30min, or until products have migrated to about half way down the gel. Ethidium bromide was used for staining.
For amps, each sample consists of 12.5ul EconoTaq, 6.5ul DNA-free water 1ul forward primer, 1ul reverse primer, and 4ul of sample. PCR is run for 40 cycles with an annealing temp of 48c.
1: Negative control (bone and DNA-free water without having going through the extraction process... water was removed and amped)
2-9: Test samples
10: PCR positive control (DNA extracted from blood, with known success)
12: PCR negative (DNA-free water instead of extraction sample)
I didn't expect anything to show up in wells 1 or 12, and the gels confirm my expectation. However, for wells 2-9, it looks like the ethidium bromide is staining something around the wells, along with a light smear continuing down each lane. What is going on here? Is it genomic DNA that isn't being amped by our primers? Our next step is to test another set of primers for a different STR and see if maybe that will amp something. Is our DNA too degraded? Is there a different angle we should maybe approach this?
Thanks for any comments/suggestions/insight. It's very much appreciated. If there is any other info you'd like me to add, just let me know.
Edited by strandr, 14 May 2015 - 10:53 AM.