I'm pretty much doing anything I can to try and fix my Gel. I'm trying to get Drosophila DNA to run through a PCR and have our products show up on a 1% agarose Gel. We're using a Ethanol Extraction to extract their DNA. Here's the protocol we used with our last extraction, PCR run, and Gel set up, as well as a picture of how the last gel turned out.
2 tubes of 4 flies and 2 tubes of 8 flies were collected. 50 uL of H2O was added and the bodies were homogenized (Or grinded, but homogenized sounds sciency). Another 150 uL of H2O was added to the tubes and were vortexed. Tubes were then placed into a 14000rpm centrifuge for 5 minutes. We transferred the supernatant to a new tube and discarded the old ones. Then we centrifuged again for another 5 minutes and transferred the supernatant to a new tube. Then we added 10% total volume of 3M sodium Acetate 6.0 pH. 2X total volume of 100% ethanol was added. We centrifuged the tubes again for 10 minutes. We then discarded the supernatant and added 500 uL of 70% ethanol. We vortexed the tubes and centrifuged them for 2 minutes. After discarding the supernatant we allowed the tubes to air dry and resuspended in 50 uL of TE buffer. This is where things get difficult for me. No matter what I do I can't get the pellet to resuspend by itself. I've shaken the tube, left in an a shaker over night, hot water baths, and vortexing. I now just take a pipette and mash up the DNA pellet untill all I see are very small chunks.
With our DNA ready we use ProMega Green MasterMix for our PCR set and run it through our PCR.
I prepare our Gel with 0.5 grams of agar and 50 mL of TAE buffer, 5 uL of Ethidium bromide and I run the gel.
If you want me to elaborate on any parts of what I just wrote, I'll be more than happy to clear it up. Thank you in advance!
also interesting note, in lane 5 I took up a visible chunk of DNA before running the PCR so I'm not sure if that affected the Gel in anyway.