Do you ask about the quantity of  PCR products that you should add to gel electropherosis wells. If so, I would suggest that you add 2-5 ul depending on the yield concentration. So, you do not need to dilute your PCR products.

I agree but you could also remove carefully  bubbles and shorten time on 15-20min before blue illuminator

do not dilute your pcr product, try making a thinner gel, with smaller wells. Then load 2-5 ul like Fawas said. btw, what is 1. pre run mode ??? i load my products and run the electroforesis for about 35 min and that's it.

good luck!

Thanks for the answer.

the precast gel 1.2% leaflet (Invitrogen gel) says the amount of DNA should be loaded into the wells should not be more than 200ng per lane in a volume of 20ul. My sample was too overloaded (it would be 680ng/ul times 20ul).

The 100bp ladder did not come out too. The result of gel is attached.

Are you certain that you are using the correct wavelength to detect your product?  See if you can borrow an actual UV light source.  Do not dilute your PCR product, just load less of it.  What loading dye did you use for your PCR samples?  I don't see a dye front on your gel either.

i just did some troubleshooting yesterday with agent selling this gel. we loaded diluted ladder 5 in 15 and 1 in 19 of dna sample. turned out to be fine. here attached. I just need to optimise PCR and probably you can suggest something. the 8 and 9 lane represent samples which we loaded later why no band coming out before - just troubleshooting. those smearing bands - what should I do?

check everything primers, samples, pcr mixture, polymerase  and do the pcr optimization again. I see ladder so gel should be fine now

Thanks Suzana, i was thinking to reduce the annealing time, number of cycles. I used the master mix so I cant think to optimise the pcr mixture and polymerase, except I will change to other brand of master mix.  the forward primer Tm is 59C and reverse Tm is 63C, I chose 60C for annealing time and 35 cycles. what do you think?

it seems ok, I usually have 35 cycles although with enough amount of DNA,30 might be enough. Maybe you might prolong initial denaturation time (I use 5min) and annealing few minutes, extension even till 10min but it also depends on how long is your gene. I would also check voltage and temperature of electrophoresis

What size is your amplicon supposed to be? Those smeary bands seem to be quite high molecular weight (well above your ladder). I assume your intended amplification product is supposed to be in the size range of the ladder. That suggests that the material you are seeing higher in the gel is not amplification product, but is the DNA template you added into the PCR reaction. You can determine if this is likely if you know how much template you added to each reaction and whether you would then expect to see it on the gel, given the amount of the reaction you loaded. I presume you measured your DNA concentration by spectrophotometry, since you report a 260/280 ratio. Did you purify the PCR product prior to measuring the concentration? If not, much of the absorbance you would be measuring in the reactions could be coming from the dNTPs amd primers. This could explain the very high apparent concentrations you measured (about 10 to 20 times higher than typical). Or did you purify AND concentrate the PCR products before measuring?

In general, you should be able to take a few µl of a PCR product, dilute to 20 µl in water or TE, add loading buffer, then directly run it on a gel to get a sense of whether the reaction worked and how well (estimate yield based on band brightness). If you want more precise quantification then you should clean it up first. This is essential for absorbance measurements. It is also desirable for fluorescence-based measurements, but if your reaction was successful, you can get a good estimate by fluorescence (e.g., Qubit or similar) without cleaning it up first; the signal in this case will be dominated by product.

Based on your second gel, it appears you got no amplification. Your suggested trouble-shooting solutions sound good. The annealing temp you mention (60 deg C) should work, but you could try even a little lower. You should test a range of temperatures starting at 5 degC below the Tm of the primers up to a few deg above to see which gives highest yield without losing specificity. Your extension time should be 15-30 sec (short product) or up to minutes as Suzana suggested (long products). 30 cycles shoudl be enough for most cases. I would not do more unless you find it is necessary.

Thanks so much for the answer.

The size of the expected band is 314bp and results were in that range. However bands looked faint and hardly saw. the bands slowly  following 300bp ladder and slowly became faint towards the end.

Yes, i diluted the PCR samples with water. PCR sample still within the purity range (800-950ng/ul in 1.98 mostly) at 11 days after reaction. i diluted 1 ul of PCR samples with 19ul with water. Bands were faint and my colleague suggested to paly with ratio next time perhaps 5ul in 19ul. not sure about that.

Thanks again

need highly polymorphic primers