no gDNA bands on AGE

[nancy_nicolas]

Hello, i'm new at agarose gel electrophoresis ... i'm wondering why i can't c a band or a smear of extracted genomic DNA
i did an 0.8% agarose TBE 1X solution , 0.5ug/ml ethidium bromid, 1x bromophenol blue, 100 volts.
i got a :
- great separation of bromophenol blue (3 colors)
- good bands of PCR products
- size marker bands
- but no genomic bands for the extracted DNA from a reference sample (saliva) i know it's large bands (20-50kb) ... but why i can't just detect its presence in the well or on the gel?

[butters]

Hi Nancy what is the concentration of your extracted gDNA?

[atin]

Nancy!
Perhaps your gDNA extraction did not work out successfully. Since you could visualize the marker and PCR products, there is no problem with the gel. You should consider revising your protocol for DNA extraction and must do it carefully!
Best of luck!

[nancy_nicolas]

well Atin the sample contains DNA for sure ( chelex extraction of saliva on a buccal swab ) i already got an STR full profile on the 3130 capillary electrophoresis ... the issue is that i got a feces sample from which i had to get an STR DNA profile so i did invitrogen extraction , PCR, Capillary electrophoresis ... as a result no DNA ... i just wanted to make sure if my sample contains DNA ( so it might be PCR inhibition) or is it degraded ... just matter of curiosity b4 trying stg else .. so i did a AGE on which i loaded:
size marker - reference sample (DNA from saliva) (5 ul and 8 ul) - blank -PCR products and feces sample
result: bands in all the wells except the blank of course and refrence sample :S
the feces sample showed a smear of small sized DNA fragments no good for PCR (anyway i did a rexetraction by changing some parameters and i got a partial DNA profile )

[nancy_nicolas]

Hey Butter ... well we don't have spectrophotometer neither real time PCR ... so i think i've loaded 2 much DNA ?

[atin]

oh! good you got your bands!
and I am just curious to know, Y are you trying to extract DNA from feces? I mean Y such an unusual source?

[nancy_nicolas]

well coz i have to compare it with a suspect DNA profile ... the feces trace was collected from the crime scene

[nancy_nicolas]

Hello butters , well i don't know since we have no spectrophotometer or real time PCR ... but its DNA from reference sample (saliva) and it's concentrated

[atin]

oh! so you are into forensic science!!
But feces is loaded with bacterial contaminants, don't you face difficulties with these contaminating genomes when you extract DNA or do STR profiling? (again, just curious to know! It seems very interesting!)