I used from High Fidelity PCR Enzyme Mix (thermo scientific:product number k0191) for PCR performing.
My PCR product is 3131bp from HEK-293 cell line DNA.
On the first day, I performed PCR test but reach to weak specific band ( at all gradient annealing:52-60˚C). There are not exist eny extra band and not exist primer dimer.
Cycling protocol for PCR product:
Initial Denaturation: 95˚C for 3 min
Denaturation: 95˚C -30 s
Annealing: 55˚C for 30 s
Extension 72 ˚C for 3.30 min.
Final Extension 72 ˚C for12 min
Number of cycle: 30
Component of any reaction:
10X High Fidelity PCR buffer with 15 mM MgCl2…….5ul
dNTP Mix (10mM)……………………………….……..1 ul
Forward primer (10pm)……………………..…….…...2.5 ul
Revers primer (10pm)…………………………...……..2.5 ul
Template DNA (46ngr/1ul)……………………………….3 ul (138 ngr/50ul reaction)
High Fidelity PCR Enzyme Mix……………………..…0.6 ul
Water, nuclease-free……………………………….….35.4 ul
On the second day, I performed PCR with the same cycling protocol but I used from 7ul (322 ngr/50ul reaction) of Template DNA in this reaction (insted of 3ul) to reach a stronger band. No Bands Were Observed.
Can you help me?
two suggestions:
1)try running same cycling conditions using 1/2 the concentration of template as you did on the first day.
2)take 1uL of the product which gave you weak bands and use this as your template(reamplification)
notes:
your starting material is of an exceptionally high concentration which may be inhibiting the reaction as evidenced by weak bands with a lower concentration of template and no bands at a higher concentration.
your working concentration of primers seems low to me. which such a high concentration of template you may be exhausting all of your primers early on.
-All the best
Your input amount of gDNA is more then enough increasing that will not help and could inhibit the PCR reaction. What is the Tm of your primers? Generally speaking a good annealing temperature is about 5 degrees C lower than your lowest primer Tm. Your annealing temperature gradient should be centered around this calculated annealing temperature. The separation of Tm between your primers should not be greater than 4 degrees C. Generally a blank gel or a faint but specific product indicates the stringency of the annealing step of the thermal cycling is too high. Finding a good annealing temperature of your primers as discussed above will greatly improve your PCR amplification.
You might want to try a cosolvent such as DMSO. I generally like to run between 1-2% in my PCR reaction. It may or may not help but will not hurt so its worth a try also. Hope you find this information helpful.
You can also try to change the concentration of the the primer and with the MgCl2 concentration.
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