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New to PCR


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3 replies to this topic

#1 Homeostasis

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Posted 13 February 2015 - 05:29 PM

Hello!

So, I've just started learning PCR and I'm having trouble. Sometimes samples come out good and other times it comes out not so good (large error bars for the RQ's). I am frustrated with myself and I'm hoping my boss has more patience. How long should it take to become reasonably good at technique such as this?

I know there are many factors that come into play with these types of experiments, I just want some advice from people with more experience before I start worrying I'm not keeping up. I've ran about 6 RT-PCR and qPCR consecutively.

Thanks!

#2 bob1

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Posted 15 February 2015 - 01:07 AM

Practice, practice, practice on your pipetting, that's the most likely cause. Do it until you can consistently pipette the volumes you require. Ensure also that your pipettes are calibrated and pay attention to ensuring that you are emptying the tips properly.

 

Try reverse pipetting if you are having difficulty with regular pipetting.



#3 phage434

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Posted 15 February 2015 - 06:33 AM

Also, make it a practice to visually check the volume by looking at the pipet tip. This is especially important for low volumes. You should just know what a microliter of volume looks like. Use master mixes whenever possible, which will increase volumes, making pipetting errors lower, and will also produce more uniform results.



#4 Homeostasis

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Posted 17 February 2015 - 12:17 PM

Ok guys! I was super careful with everything, especially pipetting. The last one came out very consistent! Thanks for the advice, I will keep on practicing.






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