Frequently Asked Questions
What happens when I place an order?
We use either DHL or FedEx for international shipping. Delivery should be within 3 weeks of placing an order or payment. You will receive a notification of an expected delivery date upon placement of the order. You will also receive a notification with the tracking number once the kit is shipped.
At what temperature should the IPA be used?
The technology can be used between 37- 42°C but it was optimised at 42°C for RapidDetector. That is why the temperature of the device is pre-set for 42°C for 20 minutes.
Can I add additional ingredients to the IPA Master Mix?
No. The ingredients in the master mix were optimised specifically to denature and amplify nucleic acids. Adding additional ingredients will affect the performance and negatively change the IPA reaction. Also, the concentration of optimised ingredients will change, preventing IPA to work as originally designed.
Can I add one primer before another oligonucleotide?
No. Both primers should be added simultaneously to kick start the reaction immediately.
Can I view the amplified products on an agarose gel?
Yes, but if you get a smear (not a clean band) on the gel, it is likely from the master mix. Like PCR, you can clean your amplified products before running on the gel.
What is the primer length required for IPA?
Like PCR, 18 bp works with IPA but up to to 35 bp works as well. It is better to try 18 – 35 bp to determine what works best for your template, but longer primers increase IPA sensitivity. As with PCR, IPA is subject to primer-dimer formation. It is possible to have primers cross reacting and giving false positives if you do not have perfectly designed primers.
Do primers affect IPA sensitivity?
Yes, but it will depend on your sensitivity goal for your assay. For example, if you are interested in 1,000 copies per reaction, then most primer pairs will be sufficient. If your goal is high sensitivity, it is better to perform systematic screening of about 15 – 20 primer pairs. The more the primers are tested, the higher the chance of primer pairs that can detect one copy of DNA.
What is the template size required for IPA?
100 – 500 bp works better with IPA but 1,000 bp works too. It is important to try 100 – 1,000 bp to determine the best template size for your assay. However, the smaller the template size (100 – 200 bp) the better the sensitivity of IPA.
Do my primers require any special or specific purification protocols?
Primer screening does not usually require a special purified oligonucleotide. Once the best primers are identified, it is better to use more purified primers if consistency is critical or required for your assay. Like in any other methods, batch-to-batch variations can affect the quality of primer synthesis and subsequently IPA reactions.
Why do I keep having false positive results?
If your no-template control shows positive, it is mainly due to contamination, especially if the positive control primers in the kit display false positive result when no template is added. Also, contamination can occur if you are handling high copies of the template (106/ µl or above) in the same workspace when carrying out IPA testing.
Like PCR, it is recommended you have a separate pre- and post-amplification workspace and keep a sterile working environment.
We suggest you start your IPA experiment using low quantities of the template (e.g. 500 copies / µl) and discourage opening the tube with a template of more than 10,000 copies / µl.
For endpoint methods like agarose gel, do apply strict contamination control measures, open reaction tubes in post amplification workspace.
To rule out, or clear contamination we suggest decontaminating workspaces with freshly prepared 10% bleach and using fresh master mix aliquots. You may need to repeat this bleach clean-up a few times before removing contamination completely.
My kit arrived at ambient temperature – will it still work?
Yes, kits are fine to be stored at ambient temperatures for 2 – 3 weeks and should still work well. You can run a control test on arrival of kit to confirm. For long term storage we suggest storage at -20˚C.
There is precipitate in my IPA Master Mix, will it still work?
Sometimes a small amount of precipitate can form at the bottom of the master mix. This will not affect IPA reactions. Vortex the tube to resuspend the precipitate before use.
Does primer melting temperature affect IPA reaction?
No, as IPA works at a constant temperature.
Does primer concentrations matter?
Yes. Just like PCR, you can try 0.2 – 0.5 µM but 0.32 µM works better.
I have not received my order, what should I do?
Kindly get in touch by emailing customerservice@isobiotech.com.au