Hello

Pipettes can be cleaned with 10% bleach. Do not use ethanol as it does not get rid of contaminating DNA. Are you sure your pipettes are the source of contamination? You should focus on other factors as well. You should also clean the working bench with 10% bleach.

Try using different places preferably different rooms for preparing the PCR mix and adding the template. As far as possible keep one set of pipettes dedicated for QPCR.

Use filter tips or autoclaved tips. Always centrifuge the reagent tubes for a short spin before opening the lid. As far as possible do not reuse the gloves. Pipetting must be done with steady hands.

I hope this will help.

Best Wishes.

Hello!!

Do you use filter tip?

I think use of filter tip can prevent contamination to certain extent, if the cause of contamination is truely pippet.

Cleaning procedures of pippet is diferrent depending on the sample type you use.

To my knowledge, to get rid of the contamination of nucleic acis,

use of sodium hypochlorite is recommended to clean the pippet.

(More than 3 %, more than 15 minutes)

UV light (30 to 60 minutes) may decrease the nucleic acid contamination to certain extent.

Best regards.

Dear Mr Malcolm Nobre, Thanks a lot for your detailed answer.

Dear Dr Hidehiko Takigawa, Thanks a lot for your answer and suggestion.

Dear Malcolm Nobre, I have one more question. How to use 10% bleach solution properly in the pipette. Spay into the pipette directly or spray in tissue paper first and then wipe??

Dear Syed Monzur Morshed

Spray 10% bleach in the tissue paper such that it is moist and not soaked and then wipe the entire pipette. As mentioned by Hidehiko Takigawa you can then keep the pipettes under UV for about 20 to 30 mins before use.

All the best.

Dear Malcolm Nobre, Thanks a lot for your detailed answer.

Syed,

Using filter tips, as Hidehiko Takigawa suggested, should exclude the pipets a source for contamination. If the the problem persists, you need to track down its source: Test all labware and all reagents if they are contaminated and produce false positives (i.e. add primers etc, but omit any mRNA/DNA). Use a different work place.

Thoroughly question all procedures around your assay (I have observed people re-using gloves for weeks for filling up pipet tip boxes, autoclaving plasticware for PCR in the same autoclave that handles the PCR waste, etc. ).

Dear Dr Wolfgang Schechinger, Thanks a lot for your suggestion. I got some insights from your writing.

We use RNA ZAP

soak a tissue with 10% bleach and clean the outside of the pipet do not enter fluids into the shaft of the pipet. Use filtertips to prevent contamination from aerosoles.

Best is to have a "clean" space where you handle your mastermix, primers, probes and another space where you handle your RNA/DNA of your samples. If possible have 1 pipet set for each space.

UV light is bad for the plastics of your pipet and will shorten the lifetime. UV is only effective close to the light and not on the shadow side of the pipet and neither on the inside.

you can use RNaseZap wipes from Invitrogen

https://www.fishersci.com/shop/products/ambion-rnase-i-zap-i-rnase-decontamination-wipes-4/p-4925089

In addition to cleaning your pipets, wipe down your work surface. If possible have a set of qPCR only pipets. I agree with Bonnie Molenaar that using filter tips is an excellent idea. Yes, they are expensive, but so are qPCR reagents.