Extracting DNA from Drosophila salivary gland squash slides

[davidallenjohnson]

I am trying unsuccessfully to isolate DNA from aceto orcein stained Drosophila salivary gland squashes that were fixed in acetic acid and a coverslip mounted with Coverbond (toluene soluble). They are old slides (about 30 years). I have soaked in toluene, then washed in alcohol:toluene, then alcohol. After that, I have tried the Pinpoint slide kit and also scraping off tissue and extracting DNA with the Promega Wizard DNA isolation kit. Is this DNA just too old and or degraded by the slide-making process and time? Any suggeestions?

[darkwng]

Hey there David,

I am not sure, where were they stored? -80C or 4C or -20 or at room temperature?

Anyways, I think you should take a look at the DNA you are extracting and see what you see on a gel. However, I bet you don't have that much so not sure if you want to run these on a gel to waste the DNA.

I would probably isolate something using what you are doing, and then try PCR on a small amount to see what you can get from a gene you are interested in.

There probably are better kits for extraction of DNA from slides....

Tell us what you find!

[davidallenjohnson]

darkwng,
Thanks for your reply. The slides were stored at room temperature, unfortunately. I didn't explain what I've been doing very well. I have been doing various DNA isolation procedures and running PCR on the extract with a primer that I know is good and getting no amplification at all. (I am running a re-amplification overnight right now on a small portion of the latest PCR reaction. I'll be ready to give up if this one is negative.) The kit I found is Pinpoint Slide extraction kit, which is really designed for parafins section slides. I'm afraid there is just nothing there.

[molecule2005]

Hey David.
Wow. That is tough....

I am not sure what you could do if you can't get any PCR amplification...

do you see any smearing on your gel when you run the PCR?
What about trying random primers to see if any DNA is there?

I would also try another kit.