Non-specific binding during PCR setup

[differentiate]

Hi everyone,

I come to you beggning for a solution to this problem.

I am doing PCR routinely on a mutant Hprt locus in mice. The mutation is a deletion. We have a construct that can recombined to restore function to the mouse hprt locus and we need to know whether the deletion is there or not.

My problem is this: I am getting non-specific banding quite frequently with this procedure. The bands are pretty consistent between repetitions and one band is only slightly smaller than the band I am searching for, making it difficult to call a positive a positive.

I have essentially been told that without solving this non-specific banding, I will be seen as a weak PCR technician and fired, despite having no trouble with other assays.

I beg of you, forum team, to help me figure out what is wrong and what the best course of action is.

The only thing I can't do is to do hot start. While I am sure this would solve the problem, I am not allowed to purchase the more costly taq to solve this problem. Other members of my lab do not have this issue with the assay, even when they do it at room temp except for when they put the master mixes on ice prior to adding taq and plateing them.

Please help!

[admin]

Hello,
Hot Start is not very good for optimization, trust me try to find access to a gradient machine. Ask other labs, I would ask and usually they don't have a problem as long as you are clean and polite.
have you tried a gradient PCR machine to look at difference annealing temperatures. See which temperature (highest) gets you cleaner PCR band with less streaking or extra bands. Don't worry about the size of the band, just try to get only 1 band.

You can also try 2-5% DMSO added to the PCR mix.

Also, MgCl2 can be optimized for PCR mixes to check increasing amounts.

I would start with a very large basic master mix, and calculate with excel how to add in DMSO and/or additional Mgcl2. Also, using the same reactions but with duplicates, I would try different temperatures for annealing. Ie if you primer annealing temperature is 57, try a gradient from 52-70 C. See which same pcr reaction gives you the cleanest result with which temperature.
You can post your picture here after PCR and gel running and I can help you select the best condition.

Good luck.

[differentiate]

Thanks so much for your advice.

I tried this technique on a gradient machine we had in lab. Didn't know about such a function.

Anyhow, tried that and tried a cycle # experiment. Settles on 64 instead of 61 degrees and 31 cycles instead of 35!

Thanks so much!