Trouble with doing DNA Isolation and then running a PCR - (Jul/25/2007 )

Hi all,

I am trying to genotype mice by isolating their tail DNA and running a PCR to amplify the gene of my interest. The Tail DNA isolation involves the use of ethanol. I tried this a couple of times earlier and it used to work perfectly, but recently, I have been seeing no DNA bands or a smear in my gel after I am done with the PCR.

I have used polymerases from Takara bio and New England Biolabs.

Recently I tried a new Tail DNA isolation technique which required the use of NAOH + EDTA and Tris-Hcl solutions, but this also did not work.

I am unable to troubleshoot. I have ordered new primers and new polymerase kit.

The working conc. of my primers is 10mM.

If someone could help me solve this. I have been trying to troubleshoot since the last 2 weeks .

Thanks in advance,

Pooja

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have you made new tris? it sounds like degradation of your template - perhaps one of your buffers has been contaminated with nucleases. EtOH will not hurt DNA, so that is not the problem - and your earlier successes were probably not a fluke, so why change methods? I would guess something has happened to one of your buffers.

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Hi, I would first make sure that you are getting good quality genomic DNA. You didn't state how you were doing this so I listed a protocol below for tail DNA isolation (just in case). I've personally used this protocol and it works fine. A Qiagen DNA isolation kit would be even better, although a little more expensive. I might check your DNA samples that you have already using a UV/Vis spectrometer to verify the 260/280 and amount. This are just approximates because you do get degraded RNA in your samples. Too much DNA in your PCR will soak up all your primers and you won't get any products. You then need to verify that your PCR conditions are good. So get a hold of a positive control for the reaction. This will let you know whether the problem is with your primer choice and PCR conditions or if it's a problem with the genomic DNA preparation. Good luck.

1. Cut 1-2 cm of tail and place it into a 1.5 ml micro centrifuge tube.

2. Add 500 µl of tail digestion buffer containing 0.5 mg/ml of proteinase K.

3. Incubate overnight at 55 C.

4. Remove the tubes from 55 C. Add 500 µl of phenol and vortex vigorously to mix the phases completely. Centrifuge for ten minutes at top speed and transfer the top 600 µl of the aqueous phase, or to just before the interface, to a fresh 1.5 ml micro centrifuge tube. Repeat this step if aqueous phase is cloudy.

5. Add 500 µl of phenol / chloroform and vortex vigorously to mix the phases completely. Centrifuge as above for ten minutes and again transfer the top 600 µl of the aqueous phase, or to just before the interface, to a fresh 1.5 ml micro centrifuge tube.

6. Add 500 µl cold of isopropyl alcohol and vortex vigorously. DNA should immediately form a stringy precipitate.

7. Centrifuge as above for ten minutes to pellet the DNA, then remove and discard as much of the supernatant as possible.

8. Rinse the pellet with .25 m l of 80% ethanol (room temperature). This step is to remove any traces of salts or phenol.

9. Air dry the pellet and resuspend the DNA in 60 µl of TE.

*You can reduce the digestion time to 4-6 hrs. by increasing the proteinase K concentration (no greater than 0.75 mg/ml) and by mixing the tails periodically during the digestion.

Tail Digestion Buffer:
100 mM NaCl
10 mM Tris, pH 7.6
25 mM EDTA, pH 8.0
0.5% SDS

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it sounds like it could be a PCR problem. I was having the same issue and turns out i needed to change my PCR settings, specifically temperature and number of cycles.

-a

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Thanks everyone for your responses,

I am trying to trouble shoot step by step. Today I am carrying out 2 different sets of reactions which also include the use of previously worked DNA samples as my controls. I am using different PCR settings and two different polymerases and bufferes to see if there is any sort of contamination. Hope it works. My hunch is I have too much DNA in my crude DNA extract and that might be the reason for no PCR products. I should hopefully know the answer by this weekend.

Thanks again,

Pooja

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would you please post a good pic of the gel, even if you don't get a discrete band?

and I don't think it's your PCR conditions, or why did it work before? another thing to consider, if you have nuclease contamination, DNA that worked OK before may not work now.

good luck, I hope you get it squared away

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Hello all,

Here are two gel pictures. I ran two different gels with two different polymerases, buffers etc. my annealing temp was 56Deg C.

Pooja

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