Hello everybody
I ran the PCR products of partial COI gene in 1% agarose gel, but its result was not satisfactory. Despite specific bands at the right place (Low Range DNA Ladder), most of them were faint. Existence a sharp band (second band in each block) shows that the primers worked properly, am I right?. Also the same result was taken by two cycling programs. Although there are primer dimers at the bottom of the gel but I ignore them.
The PCR cocktail recipe for 30 ul master mix B was:
buffer (10x truestart) 3ul
MgCl2 (25mM) 3.9ul
BSA (20 mg/ml) 1ul
dNTP's (10mM) 1ul
PrimerF (100pmol/ul) 1ul
PrimerR (100pmol/ul) 1ul
Taq poly (true start hot strat 5u/ul, 500u) 0.3 ul
dH2O 17ul
DNA template (freshly extracted) 1.8ul
The cycler (Eppendorf nexus gradient) was programmed for
TOUCH UP PCR (four left blocks)
TU: 94(4min),[94(30s),47(30s),72(1min)]10x, [94(30s),50(30s),72(1min)]25x, 72(5min)
and
TOUCH UP GRADIENT PCR (four right block):
TU gradient:94(4min), [[94(30s),47+0.5 in each cycle(30s),72(1min)]10x]5x, 72(5 min)
What should I do to improve band signal?
Thank you in advanced
Hi Kiavash
it's better to talk around concentration or quantity than in volume when making PCR.
it seems you have smears too. on the other hand, did you test your primers? it depends on the species you're working on, to see specificity, just test them on NCBI with the Primer-blast tool, or on UCSC with in-silico PCR tool.
fred
Dear Frederic Lepretre
Thanks for mentioning, I added the concentration of components. yes, the primers are specific to those taxa.
ok, you should therefore test some DMSO at 5% of the volume and decrease the Tm for 1 or 2 degrees.
fred
I'm assuming that all of these reactions were using the same primers and that each reaction has a unique DNA sample? If that's the case, then here are my thoughts:
The bright bands mean that all of your reagents are working and can amplify your COI gene. The issue is variation in your DNA. Faint bands can mean you have very little DNA or that your DNA has too many PCR--inhibiting compounds. I'd try two strategies:
First, make serial dilutions of the DNA that gave you faint bands. Try 1:10 and 1:100 and use 1.8 ul of those in PCR. Diluting the DNA will dilute out any inhibitors and can often give you stronger bands.
Second, try adding more DNA to each reaction. Double the DNA volume and add less water.
Final, seems like a trivial question, but did you add your enzyme to a shared large Master Mix, vortex to thoroughly mix, and distribute it amount all your reactions? That will ensure consistency between your reactions.
Let us know what you do next. Good luck!
Dear Katie A Burnette
Thanks for your consideration.
Yes, I made a large homogenized master mix.
According to the second strategy, I ran the reaction again (4 ul template) but the result did not change. ):
OK, then I'd say you should try diluting out the DNA and trying again. It seems counter-intuitive, but sometimes you get better results with less template.
Dear Katie A Burnette
I got confused, that is the result of the PCR according to given suggestions. There are only a few sharp bands that belong to different species, but in most lanes there is nothing.
would I get a better result if I did a second PCR round with a pcr product as a template? what is your view?
Regards
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