First when I did PCR with a new primers I saw many bands on agarose. I adjusted annealing temperature for this PCR process, 50 degree.
Subsequently, I prepared new PCR mixture from new cDNAs that I used in the previous. I decided the increase my annealing degree. I adjusted 60 degree. So only one in my samples had a band which I want.
So, I thought the doing again PCR without new preparation through new cDNA. I adjusted at this time 55 degree.. So I thought there is no substance in PCR mix to break down.
Firstly, Taq is stable for heat up to 95 degree or more than that I don't know. Moreover Taq is an enzyme, so it works everytime as long as be stable that I thought.
Second, if primer became a dimer in the same PCR tubes, I thought when the Thermal Cycler is heated up to 95, definitely if there is a dimer in my PCR tubes, is seperated that I thought.
So the rest of PCR mixture ingredients Mg+2, K+, and dNTPs that I use.
Briefly, what is the problem? Why couldn't I see any bands after doing PCR by using the same PCR samples ?
Thanks much for very important knowledge. Although I searched on the internet about tag, I hadn't found knowledge that I wanted. I couldn't taking into account the half-life OF Taq. Thank you again Mr. Yazun.
Best,
Ediz
Hi Ediz,
As Yazun mentioned, Taq could be an issue. Also, I would do a gradient PCR to verify if the annealing is the problem. These days almost every thermal cycler has the gradient option. In a gradient PCR you run sam reaction in multiple tubes, each with a different annealing temp. only. This helps in determining the optimum annealing temp. Since you used different annealing temp during the 3rd reaction (50C instead of 60C), I would recommend you verify this as well.
Gd luck!
Since you were using new primers you may need to optimalise reaction conditions. To do so,, you may need to run several PCRs using the same cDNA template testing the reaction parameters:
1. annealing templerature might be the issue as mentioned by Sumit in his answer. Gradient couls start from 50C going up to 65C.
2. concentration of MgCl2. This can be from 1.5 mM to 3.5 mM.
3. SOme GC or AT rich templates are dificult to amplify in standard buffers. Addtitives such as DMSO should be considered.
4. Touch down PCR. This means that your annealing temperature will gradually increase from the beginning.
5. The initial denaturation interval could be decreased to 2 mins. The denaturation step could be in cycling program could be decreased to 15s. This will slightly increase the amount of active enzyme. However, I do not think that the degradation of any of the PCR components is the cause of PCR failour in your experiments. In suffcient optimalisation of reaction conditions is more likely the cause.
6. Make sure that the primers are of suffcient quality and purity. Check.the vendor supplying the oligos
7. If all fails you may need to redesign the primers. The PRIMER BLAST program at the GenBank server works fine,
Best.
ÁK
I figured out my Pcr problem and nowadays I visualize a good result hopefully.
So I attain the good result for my new primers on the 55 degree but with 'new cDNAs' :)
In your suggestions in particular, thanks for 3. and 5. suggestion. I new heard about the DMSO in Pcr tubes.
Thanks all the assistance Mr,Ales.
Best,
Ediz
Mr.Ales something stucks in my mind.
I usually prepare 20ul for PCR mix and use 2 ul cDNA So, how much microliter should I add into PCR tubes as a optimal ? Do you use every time DMSO as you are doing PCR ? And there is any detrimental effect of DMSO in PCR ?
Dear Ediz,
The volume of a cDNA template in the PCR mix should not play a major role provided that the cDNA is dissolved in distilled water and provided the concentration of other components is maintained. We routinely add 4 ul of diluted cDNA into a 15 ul real time PCR mix.
As for DMSO. In most reactions, this additive is not needed. However, DMSO up to 5% may increase yield and reduce a PCR bias on some templates. One has to bear in mind that DMSO decreases annealing temperature slightly. In many commercial master mixes DMSO or other chaotropic salt in already included.
Your cdna ratios in your Pcr mix too much according to mine. I will try again with new ratio cDNA approximately like yours. And ıf I don't have a good result I will try with DMSO. Thanks :)
Have a good experience.
Ediz.
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