Tm is not the same as annealing temperature (Ta). Put your primers in an online calculator https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html?ef_id=Cj0KCQjw2ou2BhCCARIsANAwM2HUxHoK3qapuaRn9AI4GQrCRBGJ7AXIXsFG2JlPStQqAX77PCU02tsaAiq5EALw_wcB:G:s&s_kwcid=AL!3652!3!560862481324!e!!g!!annealing%20temperature%20calculator!11329799891!107921846101&cid=bid_mol_pch_r01_co_cp1358_pjt0000_bid00000_0se_gaw_nt_pur_con&gad_source=1&gbraid=0AAAAADxi_GQpDawuS2C7H7rDwf_6QLT-F&gclid=Cj0KCQjw2ou2BhCCARIsANAwM2HUxHoK3qapuaRn9AI4GQrCRBGJ7AXIXsFG2JlPStQqAX77PCU02tsaAiq5EALw_wcB#gad_source=1

and see what you get.

Also, make sure you include a positive control (if possible) and always include a negative control.

Good luck!

Can you please provide some more information what you are trying to amplify. If you are using genomic DNA as template, at least to me, this looks like that you are using too much input DNA and that your genomic DNA might contain RNA.

I am using cDNA which I have prepared from 5ug of RNA. Sabine Strehl

In which volume is your cDNA and how much are you using as input?

You need to provide more details of your PCR set-up.

cDNA is 2ul from 20ul reaction mixture, and it to 10ul PCR reaction mixture, where Forward and reverse primer is 1um final concentration, DEPC water 1ul, PCR master mix 5ul, cDNA 2ul (approx 600ng).

Thermal cycle as follows..

95c for 10 min(cycle 0f 40 with 95c of 30sec,52-63c of 40sec, 72c of 50sec) 72c 8min Sabine Strehl

There are several reasons why you might not be observing PCR bands, even though you have followed the protocol and used the correct reagents. Here are some common reasons based on the provided references: 1. **Insufficient Template DNA**: If the amount of DNA template used in the PCR reaction is too low, there may not be enough DNA for the primers to amplify, resulting in no visible bands on the gel . 2. **Degraded Template DNA**: DNA degradation can occur due to improper storage or handling, which can lead to the failure of PCR reactions . 3. **Inactive or Incorrect Enzyme**: The use of inactive or incorrect amounts of Taq DNA polymerase can prevent amplification. It's crucial to ensure that the enzyme is active and the correct amount is used . 4. **Incorrect PCR Cycling Conditions**: Using too few PCR cycles or incorrect temperatures for denaturation, annealing, and extension can lead to insufficient amplification or no amplification at all . 5. **Inappropriate PCR Buffer**: The choice of PCR buffer can significantly affect the reaction. Using a buffer that is not suitable for the specific DNA template can lead to poor amplification . 6. **Inadequate or Degraded Primers**: The quality and concentration of the primers are crucial. Poor quality or incorrect concentrations of primers can lead to no amplification . 7. **Overloading the PCR Reaction**: Adding too much DNA template can inhibit the PCR reaction, leading to no visible bands . 8. **Problems with Gel Electrophoresis**: Issues with the gel electrophoresis setup, such as incorrect staining, insufficient DNA in the gel, or problems with the imaging system, can also prevent the visualization of bands . To troubleshoot this issue, you can start by checking the quality and concentration of your DNA template, ensuring that your primers are of good quality and correctly designed, and verifying that your PCR cycling conditions are appropriate. Additionally, consider the possibility of contamination or degradation of reagents and ensure that all equipment is functioning correctly.

Thank You for your answer.

I am using PCR master mix of thermo scientific only, should I also added DMSO or DTT or any other buffer in it. I found the band of housekepping genes so reagent is in good condition.

My concern is that which formula of Tm calculation should I follow. In data sheet, provided Tm and IDT website Tm is different. Which Tm should one follow.

lastly, when I checked cDNA concentration in nanodrop it shows approx 2800ng/ul in dsDNA and approx 1700ng/ul in ssDNA. Which concentration is accurate Othman Mueen Mohammed

If you are using a PCR master mix from Thermo Scientific and have successfully amplified housekeeping genes, indicating that the reagents are in good condition, you typically do not need to add DMSO, DTT, or any other buffer unless you are facing specific issues such as primer-dimer formation, non-specific amplification, or if the protocol suggests it for your specific application. Regarding the Tm (melting temperature) calculation, the discrepancy between the values provided by the datasheet and the IDT website could be due to different calculation methods or assumptions about the reaction conditions. The most accurate Tm to follow would be the one that is experimentally validated for your specific primers and PCR conditions. If you have not done this, you can start with the Tm provided by the manufacturer and adjust if necessary based on your results. If the PCR is working well with the current Tm, there may be no need to change it. For the cDNA concentration measured by a Nanodrop, the accurate concentration to consider is the one for dsDNA, which in your case is approximately 2800 ng/µl. The ssDNA measurement is not relevant for PCR, as PCR typically uses dsDNA as a template. The dsDNA concentration is the one you should use for calculating the amount of template to add to your PCR reactions.

Sudipta Sarkar

I would suggest using this tool to determine the Tm of your primers

https://www.ncbi.nlm.nih.gov/tools/primer-blast/

And try to use less template 5µg RNA in 20µl RT is at the upper limit; and using 2µl of this cDNA in a 10µl RT-PCR reaction is a lot. Dilute an aliquot of your cDNA 1:5 with water and then use 2-5µl diluted cDNA in your RT-PCR.

If the smear in your reactions still persists and the sequence you are trying to amplify is GC-rich, you may add 2-5% DMSO.

Good luck.