I am working with Internal transcribed spacer (ITS) region primers for identification of some fungus. But I am getting some problems during amplification. So if anybody can suggest me in this regard, it will be very helpful for me.
Please explain more elaborately what kind of problem you are actually facing.
Actually I am trying to identify some unknown fungal and protozoan species. I am using the ITS primer with 52 C annealing and the DNA concentration is about 30 ng/µl. This is for the first time I am using this primer, But I do not know why it is not amplifying. I have gone through some papers but I want some specific information regarding the PCR condition. Whether I am doing any mistakes or I am missing something.
Please let me know if you have any idea in this regard.
Thank you for your interest.
If possible, please provide your complete thermal profile & also the PCR reaction composition that you are actually using during ITS PCR.
Regarding primer, have you checked online the specificity and other properties of the primers?
My pcr mixture condition is 10X- 2.5, dNTP- 0.5, Fp-0.5, Rp-0.5 and Taq- 0.25
My reaction condition is 95-1 min, 52- 1 min, 72-1.30 sec X 35 cycle.
yes I have checked all the details in internet.
Total reaction volume???
Strength of dNTPs stock?
Working Primer concentration?
Strength of Taq polymerase you are using?
Your desired fragment size?
Reaction volume- 25 µl, dNTP-2mM, primer- 10 pmol,Taq -5U and desired size 1-1.2 kbp
OK. Thanks. Please check the following,
1a. dNTPs : From this info what I can understand is that you are using the dNTPs stock of 2mM and from that stock you are adding 0.5µl in a 25µl reaction. Right? Now, if this is true, then I'm afraid to say that you'll never get your PCR product. Because, your dNTPs are becoming a limiting factor. Instead of using 2mM stock, you better use a 10mM stock and from that you add 2.5µl, so the final concentration would be of 1mM.
1b. dNTPs : If you are trying to say that your final dNTPs concentration in the 25µl reaction mixture is 2mM, then I'd say you are actually wasting your dNTPs. Instead of giving 2mM, you can only maintain final concentration of 1mM.
2. Primers : You said that you are using 10picomoles of primers and from that you are adding 0.5µl in 25µl reaction. So, the final concentration is becoming as 0.2pmoles/µl. Now I'd suggest, as you are going for 1st hand PCR of ITS, better you maintain a final primer concentration of 1pmoles/µl. So, for that, instead of adding 0.5µl, you add 2.5µl just to make sure that your primer is not becoming the limiting factor. If you get your desired band, after that you can standardize your primer concentration.
3. Thermal Profile: Instead of 1min30sec, it'd be better if you give the extention time for 2 minutes and a final extension for 10minutes. Initial denaturation time you may keep for 4minutes. Number of cycle is OK.
4. Other PCR components: The amount of enzyme you are actually using is 1.25U. It is fine. However, you may use only 1U of enzyme. For that you have to give 0.2µl.
Apart from these, sometimes you may have to add some additives like MgCl2 (final concentration from 1mM-2.5mM), DMSO (final concentration 1% - 6%), BSA etc. However, the exact concentration of these components you have to find out experimentally. Generally, in some of my cases I found that 1.5mM (final concentration) of MgCl2 and 1% (final concentration) DMSO works fine; but you have to standardize this for your cases.
5. Please check the quality of your template DNA by checking the A230, A280, A260, A260/A230 & A260/A280 values and also the integrity of your template DNA.
6. Please make sure that all the pipettmans that you are using are properly calibrated. This would reduce the chances of pipetting error. Carry out the PCR reaction very carefully to nullify the human error.
Hope, this will help. If you face any problem, feel free to ask.
please I need the primer sequences used for the amlpification of ITS region of Salmonella enterica. I want to amplify the whole region.
- Badges
- Science method