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 a PCR test but reached a weak specific band. There does not exist any extra band or 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 obtain a stronger band. No Bands Were Observed.
Can you help me?
Your annealing temperature is 95 C?? Are you sure? Annealing is usually 50-60 C... You could try a gradient temperature PCR if your primers are not annealing. :)
you probably overloaded your reaction with DNA, sometimes more does not mean better! DNA can inhibit PCR if you put to much and in some cases loading less DNA template will actually yield more product. you just need optimize concentration of the DNA template for better result.
Hi, your annealing temperature looks very high. you better can check the temperature. Gradient PCR will give you better idea about the optimum annealing temp as mentioned by Sarah above. You may also change the DNA concentration. starting with 200 ng/50 ul. you can vary the concentration from 50-500 ng/50 ul reaction volume.
Hi, I had a similar problem...for a 3kb band. As has been said more DNA might inhibit the reaction.
Try a long PCR cycling protocol. You run the first ten cycles for a defined period after which you increase the cycling time for around 5-10 secs per cycle.
I had used the long PCR enzyme from MBI Fermentas (currently with Thermoscientific) and it gave me very good bands.
The number of cycles you use for the increase and the time of increase depends on your amplicon size.
Chk this link for the protocol and standardize according to your needs.
http://www.thermoscientificbio.com/uploadedFiles/Resources/prod-info-k0181-long-pcr-enzyme-mix.pdf
Hope this helped. Goodluck
You should reduce the annealing temperature at 60-62°C and should increase the cycles at 40-45. Moreover you should use 50 ng DNA (18 ul.)
I agree with Sarah. You will need to optimize your PCR reaction by doing a temperature gradient optimization, usually ranging from 55C to 66C. That way you will get an idea of the right temperature to amplify your products. Alternatively, you may want to try a touchdown PCR to make sure your primers only amplify the sequences you want.
A few suggestions.
The addition of more template wouldnt inhibit the reaction as you havent added a ton more. PCR will work from pg up to 1 ug so you are well in range.
Your annealing temp is quite low at 55 the higher you can get it the more specific your reaction will be. Try and design your primers in fture to anneal at 60 if you can. As others have suggested an annealing temp gradient should give you the optimum temp.
Optimal primer conc in most cases is 0.2 uM you are using 0.5 uM
Your extension time is very long considering you are using high fidelity taq a general rule of thumb with such rapid processing taq is usually 20 - 30 seconds per 1kb coverage so approx 1.5 min for your product size. Normal taq 1 min/Kb.
May sound stupid but are you absolutely sure you added all reagents, is there a chance you missed something?
Really PCR is 10% design and 90% voodoo. Sometime you get primers that will work no matter what you throw at them and other times you can spend a week getting them to work, to find 2 weeks later they stop working.
Generally less is always more in PCR.
I agree with Robin, your DNA concentration is ok. You should increase to 40 cycles.
Could it be that your primers bind in high GC% regions? What is the calculated Tm of your Primers at 1.5mM MgCl2? If it is really high, than you can test different MgCl2 concentrations or you can add different DMSO concentrations (
I agree with Olga Novikova. You have to optimize DNA concentration. Try, also, to reduce the extension time. You don't need 1min per 1Kb. I'm using 30 sec extension time for a 1.3 Kb PCR product and the results are perfect.
I hope the best
Just a thought, it is very concerning when researcher states that 'Really PCR is 10% design and 90% voodoo.' Honestly, there is no place for 'voodoo' thinking in science...
You could perform a gradient PCR to know the best annealling temperature.
Hi Mohammad.
What do you need your PCR product for? Why do you want to get more? If you are going to clone it, even weak band is enough to clone it into TA PCR cloning vector like pGEM-T. Later you can subclone your fragment wherever you need.
I am using High Fidelity mixture for long time and did not read their last manual , but in former time they recommended to decrease extension temperature to 68C and increase extension time (ca. 0.5 kb/min) for templates longer then 3 kb. By my experience it is very important point. Sometimes it helps a lot to successfully get problematic PCR products with even shorter length. I would try it.
Increasing number of cycles helps only a bit and very dangerous in terms of mistake, because Pfu is dying much faster than Tag.
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