The fastest and cheapest method is without a doubt the "hotshot". However the "extracted" DNA is often of questionnable quality. If your PCR works with hotshot samples then do it this way (although I doubt that the quality will be sufficient for RFLP analysis). If not, I suggest a standard phenol:chloroform as the 2nd cheapest way (most solutions can be prepared in lab, thus reducing the costs). If it is properly done, it's also very reliable.

Thank you Sir,

We are planning to try the hotshot method, as you said, we have used the phenol-chloroform method and it is working fine, the use of phenol and chloroform is the only problem. I stumbled upon a recent article which uses only Tris-HCl and Proteinase K. I am attaching the article. What is your opinion about this method?

While I tried every other methods, I never saw this one. In their case it seemed to work great. It's definitely worth trying it!

Thank you, sir!

My lab uses both Proteinase K and the Hotshot methods of DNA extraction for PCR on mouse tails. Our default method that we always use is Proteinase K as it gives us highly reliable and very good DNA extraction resulting in perfect gels almost 95% of the time. On the occasion that Proteinase K does not work we end up using the Hotshot method.

@Josiah Fernandez.....Sir, Did you use only Proteinase K and Tris-HCl for DNA extraction as described in the article (Sysol et al. 2013), (please see the pdf article pasted above in this discussion thread)?

We use a combination of Proteinase K along with a solution from Viagen called Direct PCR (Tail) (http://www.viagenbiotech.com/index.php/directpcr-lysis-reagent-mouse-tail/tail/500-mouse-tails-100-ml.html). It makes it easier using that instead of making up separate batches of Tris-HCL.

Specifically our Proteinase K is at a concentration of 20 mg/mL and we add 10 uL of Proteinase K per 1 mL of the DirectPCR (Tail) solution. Once we have the lysis solution made up we add 300 uL per tail. For example if you have 45 tails (x300 uL each) you get a total volume of 13,500 uL. Simply round up to the next whole mL to make your calculations easier and to account for any pipetting error, so you would go up to 14 mL. Simply dispense out into each tube and place in a 55C heat block or hybridization oven which can be left overnight. The following morning just bring the temperature up to 85C for 45 minutes - 1 hour and allow to cool for 10 minutes afterward. Spin down and you're good to go. Hope this information helps.

Okay.......Thank you for the detailed procedure. I will check out the distributor of Viagen Biotech in India. If it is available, I can go for your method also. We need to standardize a method for we will be routinely doing the genotyping in future.

@Jugajyoti Baruah

did you measure the DNA concentration and purity of your extracted DNA?

we used QIAgen tissue DNA mini kit 250 (cat. no.51306) for extraction from mouse tail, we have done it according to manufacturer's instructions. DNA yield was fairly good with reasonable quality. we did PCR-RFLP using the extracted DNA and got desired results. The kit was okay, but the procedure involves many tube changes and a bit time consuming if you are dealing a large number of samples. Also expensive (as far as Indian standards are concerned) as the kit costs around 50,000 for 250 samples.

we measured the concentration and purity of DNA extracted using Nanodrop and found that,

concentration - ranges from 20 ng per microlitre to 150 ng per microlitre. most of the samples had a concentration around 30 ng per microlitre.

most samples were pure with an OD260/280 between 1.8 and 1.9

I had to dilute my DNA template for getting PCR. I used a dilution of 1:5 or 1:10 for PCR DNA template. if your sample have around 5 ng/microlitre of DNA, you will get PCR. Samples should be pure, if you are trying restriction enzyme digestion. In our case it turned out good.

hope this helps, good luck for your research.

@Jugajyoti Baruah, please read the above comments of this thread.... Josiah Fernandez had outlined an easy method, i have also pasted an article for easy extraction of DNA from tail using very few reagents. I cannot vouch for the quality of the extracted DNA as I haven't tried that method yet.

Hi Ramachandranpillai, I am also looking for a good alternate to Roche genomic DNA purification kit, primarily because of high cost per sample. We genotype by HRM which is a very sensitive technique so ordinary purification cannot replace Roche purified samples. I am wondering if you have found a good alternative protocol by now.