qPCR - No or Low Signal of Internal positive control

[Molven]

Hi,

When I perform qPCR the internal positive control of the reactions from 1-5 show low or no signals,

compared to the rest of the reactions (approx. 20 reactions are performed in one run). PCR runs were performed by colleagues 

several times with the same material and everything went well. 

I am not sure what is going wrong and I dont know why the first reactions are affected and the other ones are fine,

I assume either pipetting  error (not enough IPC) or inhibition of IPC.

I am thankful for any advice.

 

[CharonY]

If the reaction is well established and the components worked in the hands of others it is quite likely pipetting error. Inhibition is unlikely if the actual target worked well. It is possible that the internal control (depending on what you use) may have degraded, but again, if the same material worked for others, it is unlikely.

[Molven]

I wonder what kind of pipetting error, maybe not enough IPC captured? inaccurate pipetting? but why always the reaction from 1 - 5?

I know its hard to tell without watching but what can I change or how can I improve my technique.

Thank you for your help.

 

[CharonY]

I have no idea what reaction 1-5 means. Without documentation these descriptors only make sense to you and you'll have to better describe what precisely you are doing and how you are doing it.

That being said, assuming that this is just a lab course, I would talk to instructors/TAs to have them take a look what you are doing. If it is part of undergrad research you need to talk to your supervisor. 

Especially if we have many students we do not necessarily know the issues that are (as we rely on reports an what the student tells us). If the reports/descriptions are superficial or based on things folks may have read on a random forum, it actually makes troubleshooting harder for us.

We generally do have certain protocols for a reason (e.g. the way we set up master mixes) a precise description of what you did (at every step, in detail) can help spot deviations and issues. 

One simple thing you can do for yourself is to use water and check how precise your volumes are using an precision scale.