I'm beginning NGS library preps in my lab and I want to do everything correctly. My question now is about different sets of pipettors for different points in the workflow. We will have a dedicated PCR workstation.
1. RNA only: Just for RNA work. Will live in the PCR workstation. Question here though is, should this set just be for pure, isolated RNA, or also for upstream processing of RNA (from tissue sample to column). This seems like a dirtier process, and I'm wondering should I have yet another set for tissue-to-RNA processing and keep outside the workstation?
2. Pre-PCR: Unamplified samples DNA and cDNA samples. Will live inside the PCR workstation.
3. Post-PCR: Amplified products from PCR. Will live outside the PCR workstation.
Any other thoughts? Do I need another set for reagents like primers and such PCR mixes and enzymes and such? I'd rather be overcautious than not enough.
Separate sets of pipettes and these places should be isolated from one another:
1: Sample preparation (separation of blood cells or serum / plasma if you work on Blood)
2. RNA extraction (from e.g. serum / plasma which is already isolated above
3. Preparation of master mix
4. Addition of RNA to master mix
5. RT-PCR lab
6. Analysis of RNA on gel or sequencing
Now, if pipettes from RNA extraction or sample preparation area are allowed to use in master mix area, then by aerosols, your pipettes will get contaminated.
If pipettes from post-PCR product area (7) if used in PCR room for the master mix, then its called as carryover contamination, which will not go away easily, and its a disaster,
I appreciate the above answer, one thing I would like to add that put your all pipettes in a separate box for separate set of works and opened in a pre-cleaned environments only.
Its simple
Hope Im not stating the obvious...
Different sets of pipettes are important but also make sure you use pipette tips with filters. I've invested in cleaning kits such as DNAzap and RNase away and set up a regular cleaning rota. Make sure different labcoats and gloves are present in each area and you avoid transferring contamination on tube racks etc. The biggest challenge is often other people in your lab not following systems because they dont understand the importance of avoiding contamination!
Have you thought about the appropriate negative (and positive) controls for your NGS work, for example batch to batch effect of using kits? See Salter et al BMC Biology 2014 for the effects of contamination in metagenomic sequencing. You can try to prevent contamination as much as possible but the system still has to be workable
I support all the above and add the following.
Different set of pipettors to set up PCR reactions and a different set for PCR products (post-PCT reaction)
Never use a set of pipettors that dealt with amplified products to set up a new reaction.
Rodrigo
Agree with Rodrigo. Also get in the habit of wiping down your area with a 5% bleach solution before you get started. This will take care of bench contaminants. Barrier tips are essential. And it also doesn't hurt to place your pipettes in a TC hood flooded with UV for 15 min. The small things will make the difference.
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