1a) Could KO1 be heterozygous? Did you expect no amplification in KO1 and KO2? Why don't you sequence the pcr bands?

1b) Don't know... never seen something like this before.

2) Sometimes when you load too much PCR product it happens to see a smear like that. Try also to be more stringent with amplification conditions. Try 2 minutes for your long amplicon if you suspect that 1.5 is too short it will not negatively affect your reaction.

Hi, thanks for suggestions

1a) I would expect them to be heterozygous because they are first generation from seeds we obtained (and they do not even guarantee it's transformed). However, that doesn't explain why there are two diferent sizes with the WT primers and why the KO1 gives 3 bands in total (2 with WT primers, one with KO primers)

2) it should be the standard amount we use always.

Actually all these samples were rn at 68°C for 2 minutes, I was rather thinking whether it could be shortened.

1a) I think that you obtained probably segregating seed (1:2:1). One hypothesis is that one of your WT primers can somehow anneal to the T-DNA insertion or that some rearrangement occurred (i.e. the shorter amplicon compared to wild type is due to a deletion even if this is unlikely). Thus in the wild type you have one band (homozygous), in the heterozygous 2 bands (1 like wild type, 1 mutated) and in the homozygous mutant (3rd lane) 1 band (the mutated allele). The band obtained using KO primers is present as expected. But everything will be explained simply sequencing those bands...

I agree, you need to sequence this fragments to get the answer. I am not specialist in knock-outs, but did you regenerate your plant from cell culture and then obtained seeds from that plant? gDNA (KO1) you have used is extracted from several seeds or from one seedling?

@Angelika - no, the seeds were purchased, we did not transform the plants by ourselves. From these seeds plants were grown, leaves collected and gDNA extracted.

Well, my thought were about somatic mosaicism in the plant regenerated from cell culture. Then likely, this will not be the correct version.

a) as you can see, the combination of primers for WT allele gave bands of two different sizes, different for WT plant and for knock-out. And in the other knock-out there is combination, which means there must be three alleles, because there is band also with the KO primers :-/

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Here are my best guesses:

Lane 1, WT: I don't know what ladder you're using so I'll make up numbers. Let's say the WT plant with WT primers produces a product that is 500. Great, everything looks as it should

Lane 2, KO1: Both the primers for the WT and the KO amplified. I would call this sample hetero.

Lane 3: KO2: Only the KO primers amplified. I would call this sample homozygous knockout.

As for the different sizes, there's no problem there unless your placeholder/null/whatever you name it sequence that was inserted into the KO (or the disrupted gene) is bigger than the gene that is being KO'd. So let's say the gene is 490. The WT primers flank the gene and give you a product of 500. Now let's pretend the KO plant has a null sequence of 500 and your expected amplicon must lie within that, yeah? So it will be slightly smaller than the WT product. If your KO primers flank a longer sequence than the WT primers than yes, something is wrong here. If not, I think you're ok. Unless you are quite sure you got two hetero samples, then there is something off with your KO2 plant.

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b) the size of the amplicons (also of those in lower part of gel) is smaller than it should be (when compared with marker). However, previously the size was OK and now the ratio is more or less also OK, so if one shifted the marker in photoshop the sizes would be fine. I have also encountered something like that previously. Do you have any idea what could have happened? Is it really just some fault of the electrophoresis or is something rather wrong with the amplicons (all of them)?

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b) IME, shifting can result from running the gel at too high an amp, not having a completely homogeneous gel (lumps, improperly melted, improperly poured, etc.). I begin with 5 minutes at very low amp/V then crank it up a bit.

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2) for another locus we've run PCR with gDNA from two knock-out plants with two different fw primers (on left and right) and one rev primer in the insertion. As you can see, for one combination of primers it worked, not so much for the other (although there is some amplification, after moving the marker (see above) of about the right size). However, there is a huge smear. What can one do to remove such a smear?

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I agree, you need to optimize. This is easiest/quickest if you can get your hands on a gradient machine. Bump the Ta up quite a bit. Make sure your samples hasn't been degraded; i.e., is it stored at the proper temperature in the proper buffer, etc.

Good luck.

@Artur

"KO and WT product sizes are not to be compared directly."

What do you mean by this?

So what would you call the KO2 plant? ko homozygous?

What's Ta? annealing temperature of the primers? we've tried that in the meanwhile and for the primer combination on left we didn't get any amplification anyway. For the primers on right we got amplification for up to 68°C (just as in the PCR on the picture).

We couldn't use shorter elongation time because of the primers on left, which should give around 1500 bp amplicon.

@Artur:

I didn't mean to compare size of the amplicons, but rather saying - with WT primers there are two bands -> 2 different alleles? with KO primers there is a band, so there must be the insertion => 3 alleles (although I do not expect that to be true, unless the plant would be polyploid. I don't know how easy it is to get such rice plants)

We have already tried different temperatures up to 72°C (by 2°C) and 68°C was the highest were we got any (specific) amplification.

OK, now I probably understand what you mean by "PCR products obtained with the same primers can be compared"

but one of the KO primers is designed for the insertion, so there should be no amplification in case of WT allele.