I have been troubleshooting a RCAG PCR reaction for a couple weeks now intermittently between other tasks. Every time I run it with my extracted DNA, I do not see any bands after I run the gel. The heterozygous and wild-type controls both worked, and have similar DNA concentrations and 260/280 and 260/230 values to my extracted DNA samples. Is there any way to tell if something could have gone wrong during the extraction to make the DNA not viable?
My DNA Extraction protocol:
1. Lysis of Mouse Tail - 495uL Lysis Buffer (1mL 5M NaCl + 0.5mL1M Tris-HCl pH8 + 2.5mL 0.5M EDTA pH8 + 2.5mL 10% SDS + 43.5mL dH20) + 5uL Proteinase K - incubate at 65C overnight
2. Centrifuge RT 3mins full speed (15223rpm)
3. Transfer 400uL from top of solution to new eppendorf
4. Add 800uL Isopropanol
5. Invert several times (can see some precipitated DNA sometimes)
6. Centrifuge full speed 4C 10 mins
7. Dump supernatant
8. Add 700uL EtOH to wash pellet
9. Centrifuge 12000rpm 4C 10 mins
10. Dump supernatant
11. Air dry pellet
12. Add 100uL TE buffer (400uL 1M Tris-HCl pH8 + 80uL 0.5M EDTA pH8 + 39.52 mL dH20)
13. Vortex and incubate 37C overnight (or > 1hr)
I would have two potential concerns here, 1) do you really have any DNA in your tail prep? and 2) Could the DNA contain something that is inhibiting the PCR reaction. For the first one, I have seen lots of problems with small DNA preparations and recommend measuring your DNA sample with the QBit fluorometer or on an agarose gel (this would also show whether it is degraded (don't confuse RNA intensity with high molecular weight DNA). Spectrophotometry works fine, but you need to make sure that you are not measuring contamination. 2) your prep looks pretty crude and may not be very clean. Assuming the lysis is complete, I don't see anything in your prep for effectively removing RNA and am uncertain that protein is fully digested and removed. http://hedricklab.ucsd.edu/Protocol/TailPreps.html has a similar preparation, but uses NaCl precipitation to remove protein and uses a lot less isopropanol to get more specificity for high molecular weight DNA precipitation.
I calculate your lysis buffer as having 0.5% SDS, .025 M EDTA, 10 mM Tris and 100 mM NaCl. That is definitely not enough to cause protein precipitation. I have never used NaCl to precipitate protein myself, but always have used phenol or phenol chloroform to remove protein.
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