DNA extraction from rodent tails - (Jan/13/2007 )

Doe anyone have experience with Qiagen DNeasy Tissue kit for isolating DNA from single mouse tails? I used it and got very little DNA......insufficient to OD at 260, or for PCR. Do you elute in 100ul or less? Is the proteinase K in the kit any good? How much do you typically get?

Thanks,

Sybr

Just wondering...
Is the mouse tail fully submerged in proteinase K?
After proteinase K incubation at 55 degree overnight, there should have no tail left and only hair left!!
I got a lot of DNA...enough to do more than 50 PCR!!!

If you got low yield, you may elute with smaller volume (say 50ul).


I hope this may help.

Thanks for your reply. The tail is fully submerged and incubated overnight. Do you use the proteinase k from the kit, or another source? Do you elute once or twice? Could it be the size of the tail....I noticed it was less than 0.5cm.
Please advice soon.

Thanks,

SYBR

I use the proteinase K from the kit...elute once.
I think you can only cut 0.5cm off the mouse tail.

Just wondering...
1. Are your kit expired?
2. After adding proteinase K to the tail, did you vortex it before putting it in the incubator?
3. Did you vortex the lysed tail before adding to the column?
4. What volume did you elute the column with?

Thanks so much for your response. I tried again and nothing! I have a very small piece of tail....0.3cm only. I eluted in 100ul of elution buffer and followe the protocol exactly. Do you suggest 50ul only? Would you process an ear punch sample the same, and elute in 50ul?

Thanks for the help,

Sybr

Just wondering,
1. Did the Buffers AL and ATL precipitates? If yes, warm them up at 55 degree.
2. Did you add ethanol to Buffers AW1 and AW2?
3. Did you freshly mix 200ul Buffer AL with 200ul ethanol per preparation every time? Although the protocol said you can keep the AL-ethanol mixture for at least three months, it is better to make fresh every time.

If the above questions are "yes" in Q2 and Q3,then I think something wrong with your kit.
You may wish to buy a new kit.

Hope this help.

Thank you. It worked nicely. I eluted in 60ul once only, and the conc and ratio are much more reasonable. The problem now is with the PCR. How much of this genomic DNA would I use. I tried 1ul and got smear only. Any ideas?

Sybr

I thought it depends on your concentration of your DNA. Quantify first, then only run for PCR. To work with PCR, I think the recommended one should be around 50ng to 100ng of DNA inside the whole PCR mix solution. Though some claimed that lower than 50ng might work as well. This shows how sensitive PCR is. =)