In problem 1 if the reagents all worked recently then the most likely cause is that the primers are dissoved in water and have degraded either due to nuclease activity or by depurination.

use new primers.

In problem 2 one likely possibility is that you forgot to add the positive control dna or put it in the wrong tube

Make primers for ActB or Gapdh. Check your primers with Primer-BLAST. A positive control would really help. Good luck!

There are several options:

1. Smth went wrong with the reagents (storage conditions, fail at production stage) - try with different PCR kit (or maybe even PCR kit by different supplier)

2. Smth went wrong with amplificator -thy another one

3. Smth went wron with electrophoresis (one day I saw no bands because of ethidium bromide did not stain the DNA in samples, while the ladder was pre-incubated with dye and was visible). Check if any DNA can be visible in the gel.

Check if gel electrophoresis is working properly

1. Gel perforated hence lost on loading

2. Gel loading dye intergrity-can simply check if ladder is loading

3. Running buffer at correct pH and concentration

Please upload photos of gel

otherwise as Paul says additionally fresh reagents have something missing-is it a commercial mastermix? Poor primer dilution too.

Try to add to your master mix DMSO or BSA, it worked for me when I was having problems with my PCR.

If you observe complete PCR failure with no bands in any lanes, but you cannot change the protocol, follow this systematic troubleshooting approach:

1. Check Template DNA

• Ensure the DNA is of good quality and free from inhibitors (e.g., EDTA, phenol, heparin).

• Verify DNA concentration with a spectrophotometer (A260/A280 ratio ~1.8 for pure DNA).

• Run the DNA on an agarose gel to confirm integrity.

2. Confirm Reagent Integrity

• Polymerase: Use a fresh aliquot of Taq or the enzyme specified in the protocol.

• dNTPs: Ensure they are not degraded; use a fresh mix if needed.

• Primers: Check for proper storage; avoid repeated freeze-thaw cycles.

• Buffer & MgCl₂: Use fresh buffer and verify Mg²⁺ concentration is correct.

3. Verify PCR Setup

• Master Mix Preparation: Double-check reagent volumes and concentrations.

• Pipetting Accuracy: Use calibrated pipettes; ensure proper mixing.

• Positive Control: Run a known working template to confirm PCR functionality.

• Negative Control: Ensure no contamination is causing unexpected results.

4. Assess PCR Cycling Conditions

• Confirm thermocycler settings match the protocol (denaturation, annealing, extension).

• Check if the lid is heated (prevents condensation).

• If using a gradient cycler, verify temperature uniformity across wells.

5. Evaluate Gel Electrophoresis Issues

• Ensure the gel is properly prepared and the right percentage of agarose is used.

• Verify the DNA ladder and loading dye are functioning correctly.

• Confirm the gel is running in the correct direction with proper voltage.

By systematically verifying each step, you can identify the issue without modifying the protocol.

Hi Sachin, here are some suggestions:

Step 1: Check if the electrophoresis system is working This one’s easy — just make a fresh gel with fresh buffer (TBE/TAE), and run a lane with only the marker, nothing else.

• If the marker looks good, then your gel, buffer, and running system are fine — move on.
• If the marker looks weird or disappears, then the issue is likely with the buffer, dye, or the gel tank itself — no need to worry about PCR until this is sorted.

Step 2: Check your DNA quality Take your extracted DNA and run it directly on a gel. Look for a nice genomic DNA band (something above 10 kb), and check the Nanodrop readings too.

• OD260/280 should be between 1.8 and 2.0.
• OD260/230 should be at least 1.8.

If the DNA band looks bad (smearing or very faint) or the OD ratios are off, there’s a good chance the problem came from the DNA extraction step — and that could definitely explain why the PCR failed.

Step 3: Double-check your PCR mix and setup

• Use fresh Taq, dNTPs, and buffer.
• Add reagents tube by tube, no master mix this time (especially when troubleshooting).
• Use fresh aliquots of primers and template — avoid old tubes that have been freeze-thawed a lot.
• Set up all the lanes properly: positive control, negative control, samples, and marker. Controls are key when things go wrong.

Bonus tips (just in case):

• If your positive control fails, try running a reaction with just water + positive control template + primers — this helps check if the reaction mix itself is the problem.
• If nothing works at all, grab a brand new positive control template and a safe bet primer set (like GAPDH), and run that — just to see if the machine and your reaction system are fundamentally working.

By the way — what kind of sample are you working with?

• Cells? Tissue? Blood? Or something more unusual?
• And what DNA extraction method are you using?

This one might be a bit off the wall but I've seen it happen. If you had LOTS of template (especially from a plasmid) you can get a band in your gel from just the template showing up.

I saw this happen when the plan was to use a positive control plasmid for gDNA test samples. The plasmid was used undiluted (2 ml of miniprep) so it showed up as a very bright control band. It was the wrong size so that was a good clue. I've also seen a PCR test of all undiluted plasmid samples so all lanes with template had bands.

So, if you have plasmid for the test samples (and something else for your positive control) you could get bands in all test samples.

It's weird but an easy fix (just dilute your plasmids)

A bit more details on the types of templates you are using for your control and your test samples would help (plasmid, gDNA, cDNA, etc.)

You may follow these steps:

1. Verify that primers, dNTPs, polymerase, and template DNA are not degraded or expired.

2. Verify the program settings (denaturation, annealing, extension times, and temperatures.

3. Confirm the gel was prepared correctly (proper agarose concentration, TAE/TBE buffer, and ethidium bromide or alternative stain). Ensure the gel was run at the correct voltage and for an appropriate duration.

4. Run a positive control (a known working template and primer set) to confirm the PCR setup is functioning.