the existence of any band in the negative control is an indication of contamination but if you want confirmation that it is your product just run a normal end point pcr and cut with any restriction enzyme that cuts your product but I would just have a major clean up of working areas and pipettes,order new primers and reagents ( or borrow all reagents from another researcher and just buy new primers) and try the pcr again

the existence of any band in the negative control is an indication of contamination but if you want confirmation that it is your product just run a normal end point pcr and cut with any restriction enzyme that cuts your product but I would just have a major clean up of working areas and pipettes,order new primers and reagents ( or borrow all reagents from another researcher and just buy new primers) and try the pcr again

Agreed with Paul. Cannot really add to his answer

Hi dear Paul and Laurence,

Thanks a lot for your comments.

Thanks for your kindness.

Best regards,

Ramin

The best way to check whether your primers are contaminated would be to set up a PCR with the same primers but using fresh aliquots of all the other PCR component reagents(ie fresh aliquots of PCR grade H2O , Taq polymerase, dNTPs, Mg2+ etc.). If you still get contamination in your PCR H2O control then it's possible your PCR primers are contaminated. However, this inference is only valid if you are absolutely certain that all the stock reagents are free from any template contamination.

It is good practice to always aliquot your PCR stock reagents into smaller volumes before using them for the first time. If a contamination problem arises you should discard all the component reagents used in the last PCR and start with fresh aliquots of all the component reagents. That way you avoid wasting time and money conducting more PCR experiments just to find out which one of your PCR reagents is contaminated.

Here are some useful tips to avoid PCR contamination.

One of the main approaches to control PCR contamination is to physically separate the working areas for:

1. Preparation of the DNA template

2. Setting up the PCR reaction mix

3. Post PCR analysis

· Many labs have a designated room for setting up the PCR reaction mix (pre-PCR room) and a separate lab for analysing the PCR products (post-PCR room). No items should enter the pre-PCR room from the post-PCR room and a separate clean lab coat should be kept for use in the pre-PCR room. If you don’t have a pre-PCR room, use a PCR hood or cell culture hood or a different part of the lab to set up the PCRs.

· Use sterile techniques and always wear fresh disposable close fitting gloves when working in the pre-PCR clean room / area.

· Before you start, clean bench surfaces and pipettes with a DNA decontamination reagent eg DNA Away.

· Aliquot all reagents used for PCR into smaller volumes for storage.

· Use a dedicated set of pipettes for aliquoting PCR reagents and setting up the PCR mix.

· Use aerosol-resistant filter pipette tips or positive-displacement tips.

· Always use PCR grade water throughout.

· Never put hands inside PCR tube bags. Always pour out the PCR tubes on a clean tissue placed on a tray or surface that has been wiped with a DNA contamination reagent.

· Keep fingers away from the top of the PCR tubes as much as possible. Open and close all tubes carefully making sure you don’t touch the inside of the caps or rim of the tubes.

· Handle PCR tubes from the bottom when arranging them on the PCR tube rack. A clean pair of forceps can be useful for this. The tube rack should be clean and wiped with a DNA decontamination reagent before you start.

· Do not splash or spray PCR samples.

· Change gloves frequently especially if contamination is suspected.

· Keep reactions and components capped whenever possible.

· Premix reagents before aliquoting into individual reactions to reduce the number of pipetting steps.

· Minimize opening of PCR tubes post-amplification. Gel electrophoresis of PCR products should be carried in a separate lab. Real-time PCR systems eliminate the need to open reaction tubes for analysis.

· Once you have finished aliquoting your PCR master mix into the PCR tubes the water should be added to the negative template control tube in the pre-PCR clean room. All reaction tubes should then be closed and taken into another lab where the DNA template is added to the rest of the PCR tubes.

Hi dear Rajai,

Thanks a lot for your comments.

Thanks for your kindness.

Best regards,

Ramin

Hi Ramin, you might also try a end point PCR with random primers using your contaminated primer as source of "template". Your 2 controls should be water and a clean source of the primer used as template before.

Here is a guide for amplification of trace amounts of DNA.

https://www.google.de/url?sa=t&source=web&rct=j&url=https://www.biotechniques.com/multimedia/archive/00009/03354st06_9908a.pdf&ved=2ahUKEwjdwYOExeTYAhUC26QKHWyVAg4QFjAAegQIFRAB&usg=AOvVaw1NPpDz14JmW3VAeM9IJnEN

Best,

Hi dear Sebastián, Thanks a lot for your comments. Thanks for your kindness. Best regards, Ramin

In addition to all the great advice already given above, if you don’t already use IR Spectroscopy (eg nanodrop), be sure to do so. Run triplicates measured both pre- and post- mixing on each of your samples, f-primers, r-primers, and of course the h2o used to adjust PCR master mixes/samples.

independently measure each one from stock supply and then repeat on each sample after you’ve normalized concentrations with h2o, which ideally is RNAse or Nuclease free.

Aside from measuring the concentrations, which you already know to do, you’re also confirming each ingredient used downstream is pure, non degraded, and free of contaminants. Saves time later on if you run into trouble bc you already verified the quality of each of the constituent parts.

Most important advice to follow though is that from Rajai above, each and every word of it. If you mistakenly omit/violate any of those steps, trash the sample you were working on and start over.

Take care!