Below is the gel image I got following a blue-white colony screening and colony PCR. I got the right bands for my inserts in lanes 2&3 but got only fuzzy, non-specific bands of roughly the same sizes in other lanes (except for lane 6, the negative control). The primers we used would produce DNA fragments of size that agrees with where the fuzzy bands are, given that there's no insert, so I thought that the PCR reaction was successful, just that the plasmids didn't have an insert for those lanes. However many of my colleagues agree that these are caused by an excess of DNA/bacteria added in the PCR that inhibited the reaction. Now my questions are:
1. if the truncated DNA fragments/fuzzy bands were caused by inhibition, how come they are all the size we'd expect for a successful PCR, just without an insert?
2. If the reaction is inhibited after the primers bind, how come we end up with short DNA fragment indicated by the gel instead of the entire vector (since no reaction happens and no short strands are made), which should be a lot larger?
3. If I'm correct that the fuzzy bands are simply caused by lack of insert, why do I have both a fuzzy band and an insert-containing band in lane 2?
Thank you all so much in advance.
It's a bit difficult to tell without knowing the size of the bands on the DNA ladder, but the fuzzy bands look like unincorporated primers/primer dimers to me.
Inhibition seems unlikely, you would have to add a lot of DNA/bacteria for it to inhibit the reaction. The reason why I think inhibition is not the case here is - if I am correct in assuming that you added roughly the same amount of DNA/bacteria in each reaction, then why should the PCR work for colonies 2 and 3, but not the rest? The fuzzy bands do look like primers to me. As for why there is an insert band as well as a fuzzy band for colony 2, you might have a mixed colony there (maybe there was a tiny blue colony too close to the one you picked?). Or it could be that you added too little DNA/bacteria there. Hope this helps!
Hello Luna
I am agree with others. It's very difficult to tell without knowing size of PCR product and ladder size. Though fuzzy bands corresponds to lower size bands of ladder, might be primer dimer. But again it can be confirmed after knowing the ladder size.
Thank you everyone for your answers! I updated the image to include the band sizes. Also, according to the semi-log plot generated from the ladder bands, lane 2's band (boxed) was calculated to be 751bp and lane 3's 985bp, both of expected sizes. The band size without an insert should be 176bp (which to me looks like where all the fuzzy bands are), as we are using Promega's pGEM-T Easy vector and T7&SP6 primers.
The primer-dimer explanation sounds reasonable to me, tho I'm still a bit confused as to how all the primer-dimers ended up with roughly the same size, where the PCR product without an insert is expected? And what made them not incorporated in the first place?
Thank you all again for your patience!
With the size standards added, primer-dimer sounds much less likely.
It seems more likely now that your agarose concentration is too low. 176 bp is near to the lower size resolution for agarose gels. I would use a 3% gel, or use a speciality agarose designed for small-fragment resolution. Acrylamide is also a possibility.
Fuzzy band may be the DNA without insert. Increase the agarose concentration and run in low voltage for longer time for better resolution.
All the best.
I'd be interested in hearing others' experience with electrophoresis of small fragments at low voltage (see Kunal's comment above). I always ran mine at 130 V. My concern is that in the time required to separate the fragments at low voltage, the bands might become fuzzy due to diffusion.
I agree with Andrew, I run my gels at about 130 V as well. In my experience, running a gel at low voltage causes a considerable amount of DNA diffusion out of the gel, so the bands end up being very faint.
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