Ladder Fine but no DNA bands please help! - (Jul/18/2008 )
Hi! I am getting desperate! I have attempted to run the same DNA (for a maize mutant) about 10 times now and have not seen ONE DNA band! My ladder always looks fabulous... so I think the gel itself is okay, and thus I suppose the running buffer should be fine?
Here is what I'm doing:
I have tried a urea extraction (used by another lab on my campus) and PCR using betaine (my gene is GC rich), and run on a 2% agarose gel with EtBr at the other lab on campus and it worked well - they were watching me the whole time
Now that I'm trying it in my own lab, it has all gone to hell!
So I tried the urea extraction of the same type of maize, and forgot the betaine before PCR, so understandably I saw nothing on the gel but the ladder. However, when I went back and ran that genomic DNA extract through our nanodrop (DNA spec) it said there was no DNA!
I also have tried a NaOH (followed by Tris buffer addition) extraction from a recent publication and when I put it through the nanodrop it DID pick up DNA at 260 nm. Plus a lot of junk. But - I just ran that extraction through PCR, and did remember the betaine haha, and ran on my EtBr gel and all I got was a ladder! Not even a big genomic dna band!
Right now I'm reusing that gel and running just 2 samples of the non-PCR original NaOH extract to see if I see any big bands/smears.... but I'm not holding my breath.
Can you help me troubleshoot? I am new to this! Do I need to give more details?
Thank you SOOOOOOOOOOO much!!!
Just looked at the latest - 2 genomic samples and a ladder - all I see is the ladder! But the nanodrop said I had DNA!!!!! I'm baffled. And sad
Also - is the gel supposed to turn clear (ie the dye goes away) after photographing under uv?
Your problem might not be with the template DNA, but with the primers, polymerase, PCR conditions, or cycling program.
Thank you HomeBrew! Possibly it is with the PCR reagents - the thermocycler and program I used is the same one used (currently) by that other lab for whom the procedure is working. But it might be the primers or polymerase... I'll check on the expiry dates of my reagents!
If I want to make sure the issue is not with the template DNA, should I be able to run it on a gel as is (with dye) out of the extraction procedure?
Thank you so much!
You should be able to see DNA on a gel if you load a sample of your extraction. Seeing this is not neccesary, however -- recall that PCR needs only vanishingly small amounts of template DNA to be successful -- amplification of ancient DNA extracted from samples that were millions of years old is proof. The DNA required by PCR to produce an amplicon can easily be of such a small quantity that it is invisible on an EtBr stained gel, so when a PCR fails repeatedly, in spite of evidence of measurable template DNA, it's time to look elsewhere for the problem.
Any number of things other than the template DNA quantity and quality can interfere -- primers can stop working due to autohydrolysis or repeated freeze thaw cycles, PCR machines can fail to do what their screens are saying they're doing, PCR mixture parameters can be wrong, the number of cycles can be insufficient, there can be contaminating substances left over from the extraction that can be inhibitory, the nature of the DNA (GC content or secondary structure) can interfere, etc.
Many of these things can be ruled out by use of a positive control -- do you have another set of primers that should produce a product from the same template DNA (it doesn't matter what this product is, we're just trying to produce a band)? If you do, and this reaction works but your main reaction doesn't, you can conclude that the machine is working, the programed cycles are sufficient, the polymerase is working, the reagent mix is OK, the template is sufficient, etc., leaving you with possible primer issues or template properties (GC content or secondary structure formation) that might be stopping your main reaction.
This is a long shot, but I would immediately try again with your DNA sample diluted 10x and 100x. I'd guess that your DNA prep (which seems difficult and sketchy to me) is full of inhibitors. Since you need little DNA for PCR, dilute the inhibitors and try again. If you truly want help, then you need to tell us EXACTLY what you are doing in excruciating detail, since otherwise you may be overlooking a crucial point. Why are you not trying this again in the other lab and carefully noting differences?
I didn't think of diluting my DNA - thank you! It does seem quite "junky" - when I ran the extracted DNA (from the naoh extraction) through the nanodrop a ton of junk did show up at the lower nm readings.
I definitely truly want help - I didn't know what details other people would need to help , sorry! My last resort is to try again at the other lab - I guess it does make sense, it is just not as feasible as I thought troubleshooting might be. I definitely need to be able to do this in my own lab but now that you mention it I should go redo it at the other, maybe there's something wrong I might be doing that they would notice.
My lab notebook isn't with me at the moment but I will come back and provide details later!
Thank you very much