Problem with DNA extraction/Agarose Gel

[emallimo]

Hello,

I'm having an (ongoing) issue with possibly my DNA extractions and gel electophoresis. Some information: I am extracting DNA from animal tissue (mouse tail clip) in order to genotype our mouse colony. More often than not when I run a gel the bands of at least half of my samples are very faint or completely absent. Recently (this past week or so) absolutely none of my samples can be visualized on the gel. I am not sure what the problem is or what I am (have been) doing wrong. I thought it was a problem with my DNA extractions because I noticed that with almost all of my samples the DNA pellet never seems to dissolve into the TE buffer. Once re-suspended I heat and shake my samples overnight at 37C, but the pellet never seems to dissolve. I tried adding more TE buffer to each of my samples but this did not seem to help.

To determine whether it was my samples or my primers I ran a PCR using the same samples but different primers then ran those samples on a gel. When I did this the bands came out as we expected them to. This made me think that maybe the problem is not with the extractions (in spite of the fact that the DNA never seems to dissolve into the TE buffer) and that I may be doing something wrong when preparing the PCR.

Both the master mix and primers are new so I now think I may not be using the right amount of DNA and/or primer for the PCR rxt. As you can imagine this is very frustrating and being that I am relatively new to all this, any advice would be super helpful...

some additional information just in case: all of the buffers I have been using (i.e., lysis buffer for tail digestion and TE for re-suspension) were made within the past month, so they are also relatively new. We run our samples on a 1.5% agarose gel stained with ethidium bromide at around 45 mV.

[luisillo]

About the extraction, it would be useful to mention which method you are using. Anyway, after the last wash with 70% etanol, how much time do you dry the DNA pellet? Because if it is too dry it becomes difficult to resuspend.

About the PCR, you should try adding more template DNA to your reaction tube. You can even make a gradient by putting different quantity of the same DNA in different tubes: 0.5x, 1x, 1.5x, 2x, etc (ie. 0.5, 1, 1.5, 2 ul, respectively). You can do this with primers, too, or even preparing them again. Be aware that high concentrations of template and/or primers can inhibit the reaction.

[emallimo]

For our extraction method we digest our tail clips in 200 ul lysis buffer+ProteinaseK (100ug/mL) overnight in a waterbath at 55C. The next morning we centrifuge the samples for 2 minutes at 13 000 rpm. we then remove the supernatant and add 200 ul of isopropanol to precipitate the DNA. The samples are then centrifuged again for 2 minutes at 13 000 rpm to pellet the DNA. Next, we remove and discard the supernatant and add 200 ul of 70% ethanol to wash the pellet, then remove and discard the ethanol. finally we air dry the pellet for a couple of minutes prior to dissolving it in TE buffer. Our protocol also says to incubate the samples in the thermomixer at 37C for 1 hr-overnight (with shaking) to completely dissolve the DNA.

As for my progress with the PCR I have tried quantifying the DNA in my samples using a spectrophotometer and found that I have DNA concentrations ranging from 14 ng/ul to 180 ng/ul. I quantified each sample in an effort to make sure that I was adding the same amount of DNA for each sample to the PCR (I added 100 ng DNA). However this did not work either. For the PCR I just mentioned, I ran a 25 ul reaction; 12.5 ul master mix, 1.5 primer (our primers are at a concentration of 10 pm/ul), 100 ng of DNA, and brought the final volume up to 25 ul using water. I'm not sure what could be wrong, we never used to quantify the DNA concentrations of our samples in the past and stuck to running our PCRs using 1 ul primer and 1 ul DNA and if this did not work we would increase the amount of primer to 1.5 ul and decrease the amount of DNA to 0.5 ul; in either case we were able to visualize bands on our gels.

Is there something really obvious that I am missing or some error that I have made and did not realize?

[luisillo]

For what you say, it might be a PCR inhibitor in the DNA you extracted. Have you prepared new TE buffer? Because I think that's the first thing you should do. Also, you could try dissolving the DNA in water (once dissolved, store at -20ºC until use, make aliquots).

Alternatively, you could add an step of purification with phenol:chloroform (1:24) before you precipitate DNA, that will help get rid of any soluble protein that might be interfering.

About your photometer readings, you can check purity by calculating 260 nm/280 nm relation. Optimal purity lies between 1.6 and 1.8. Some photometers automatically give you this relation, be sure to check it.

Hope this help you solve your problem.

[knighbri]

Also, instead of making new TE buffer you could just check the pH (dna dissolves best pH of 8). Also, did you design the primers you are using or did you get them from a source where they are known to target the specific gene of interest. Even the best designed primers sometimes don't work for some unknown reason.

~knighbri