DNA extraction and PCR problems from bone

[trionyx]

Hello,
I was able to extract DNA from bone of a turtle species and see a week DNA band comparison to muscle tissue when I run the DNA products on the gel. I used Kalmar et al.,2000 protocol;
- Extraction buffer (0.1 M EDTA, 0.5% N-laurylsarcosinate, 100 mg/ml Proteinase K)
- 3.5 µl (1 µg/µl) Dextran Blue
- 250 µl 4 M NH4-acetate
- 500 µl EtOH
- 25 µl dH2O

I performed PCR of mtDNA gene(app. 850bp) to be optimized previously. But I didn’t have amplification. I used in amplification;
- 0.6 µl Primer F and R
- 2 µl dNTP (2.5 mM)
- 1.5 µl Bovin Serum Albumine (BSA) (4 µg/ml)
- 0.4 µl Taq poly.
- 6 µl DNA (uncertain DNA quantitative)

I will be happy if you help me for both PCR and DNA extraction.
Thank you…

[Shraddha Bhullar]

As per your protocol, may I ask you one question?
Didn't you use buffer and MgCl2 in your reaction mixture?

[trionyx]

I didn't write buffer and MgCl2 I used. I use 2.5 µl/per 25 µl reaction from 10X complete buffer.

[Shraddha Bhullar]

Hi,
You should try with different primers.
There could be possibility of presence of PCR inhibitors. Try to optimize DNA extraction protocol.

[butters]

pcr inhibitors can be check by including a know template and fully working primer pair in with the reaction you use. if that does not amplify then maybe it is due to inhibitors. since its mtDNA you cannot extract from other easy to obtain source? like fresh or blood?

u may need to quantify the amount and purity of ur extracted sample too. It may help to control the quality and concentration of DNA. Sometime too much DNA will inhibit PCR

[Aga]

As Butters said - you may have to check the quantity and purity of your DNA. If there's nothing - try to change something in your extraction protocol. I don't actually know how long do you incubate the samples during DNA extraction. You said there is a weak band on a gel so you have something but maybe too little.

If you have DNA after extraction the easiest way to get rid of high concentration of inhibitors is to dilute the sample. Have you tried your assay on other tissues? I worked with mtDNA and there is plenty of it in muscles - muscle cells need energy to work so there are many mitochondria.

Before doing PCR you need to have relatively pure DNA to optimize the reaction first. I don't suspect high amounts of mtDNA in bones. But if you extract some of DNA (most probably total DNA) from bones maybe the best solution would be to run nested PCR?

[vituljain]

can any body tell why dna has thymine in place of uracil ?
plz do tell me

[vituljain]

can any body tell why dna has thymine in place of uracil ?
plz do tell me

[butters]

as far as i know uracil only available in RNA. correct me if i am wrong.

[luisillo]

Originally Posted by vituljain

can any body tell why dna has thymine in place of uracil ?
plz do tell me

It's basically a matter of stability. Thymine has its ring methylated. That methyl group confers more stability (less reactivity) to the molecule. This is pretty convinient since DNA acts as an "archive" of information and needs to be fidely conserved.

Another reason is the repair mechanisms of DNA replication: when a uracil is found in DNA it is converted to citosine by adding an amino group. This is why uracil normally never shows up in DNA, and cells most avoid this at all costs since it would mean a substitution of T for C.