Your gels look like you have a large number of additional bands that are probably non-specific, so it is possible that something is decreasing the specificity of the reaction.

Could you please post your PCR recipe?

Try checking that you are using the correct Mg2+ concentration. I once had all of my PCR reactions fail for three weeks and it turned out that I had picked up the wrong tube of Mg2+ buffer to make up my mastermixes with. Even though I thought that I was making the solutions correctly, because the Mg2+ tube I had was 5x higher in concentration than what I thought I had, it completely inhibited the reaction. In your case it would probably be a lower Mg2+, rather than higher.

Another possibility is the thermocycler. If your annealing temperature is too low, this could also cause non-specific products.

• Are you using the same machine as your Post-Doc? Unless your thermal blocks are calibrated, there is likely to be variation between instruments.
• Are your cycling parameters identical to the ones that are written on the protocol? (and the parameters that the Post-Doc actually uses, in case they are different.)
• Are you placing your tubes in the middle of the thermal block or on the edge? There is usually a large amount of variation between the temperature in the middle and at the edge.

Your gels look like you have a large number of additional bands that are probably non-specific, so it is possible that something is decreasing the specificity of the reaction.

Could you please post your PCR recipe?

Try checking that you are using the correct Mg2+ concentration. I once had all of my PCR reactions fail for three weeks and it turned out that I had picked up the wrong tube of Mg2+ buffer to make up my mastermixes with. Even though I thought that I was making the solutions correctly, because the Mg2+ tube I had was 5x higher in concentration than what I thought I had, it completely inhibited the reaction. In your case it would probably be a lower Mg2+, rather than higher.

Another possibility is the thermocycler. If your annealing temperature is too low, this could also cause non-specific products.

• Are you using the same machine as your Post-Doc? Unless your thermal blocks are calibrated, there is likely to be variation between instruments.
• Are your cycling parameters identical to the ones that are written on the protocol? (and the parameters that the Post-Doc actually uses, in case they are different.)
• Are you placing your tubes in the middle of the thermal block or on the edge? There is usually a large amount of variation between the temperature in the middle and at the edge.

Hi Michael.  I have had this problem before as well.  The loading dye issue is a simple fix, just dilute it with water and glycerol or use less for each lane.  As for the PCR, you are likely running it at too low an annealing temperature in the PCR machine.  That would be the first thing I would check.  Salt and Mg2+ concentrations in your PCR buffer could also be an issue.  If it were me, I would redesign the primers.  Any primers that have that much non-specific product are not the best primers to start off with.  Good luck!

Everything works for the post-doc, she gave me aliquots of all the samples she used and we share the same machine. I do not expect any answer, I really opened this thread to document the solution, even though I expect it to work inexplicably.

Hi Michael, lot of things have been said about annealing temp, MgCl2 concentration etc... I'm gonna try to find something different. My suggestion is to order a new batch of primers (or use a different one, I don't say a new dilution of the same primers ).This one could be spoiled, if your primers are partially degraded (then shorter) they can match unexpected targets, and you get this kind of RAPD profile... Good luck

Hii Michael, David Bru's suggestion could be best solution. Since I see a streaking pattern, I feel that you are adding too much template. So you can also try with different amounts of template. For example 0.5ul, 1.0ul, 1.5ul etc. Good luck..

Did you try to use the same exact reagents, primers and template just like those that was used by the post-doc? Is it the same recipe (amount of reagents, cycling temperatures and times)? Including the same set of pipettes too? Also at the same bench and using the same thermocycler.

In that way you could at least minimise the possibility that it is due to the reagents or equipments.

The problem seems to have been the pipettes.  Somehow, they were continuously being contaminated.  The reason why is still unclear since I am not using them in any extraordinary way, and they have not been so sensitive before.  Nevertheless, I washed the pipettes thoroughly with a 3% sodium hypochlorite solution (Bleach), for 10 minutes and then soaked them in pure H2O for another 10 minutes, rinsed again directly from the dispenser, and then left to air dry. 

Now, every time before and after I run a PCR, I wash my pipettes with "RNAse Away" solution.  For the last 5 PCRs things have been clean, although I am still getting some inexplicable streaks and banding.  There have not been bands lining up with the positive control and, thankfully, these are not being used for publication, so I am able to accurately genotype my animals.

Good to hear you sorted it out Michael.

Good luck with the rest of your project!