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Our lab uses old Gilson Pipetman pipettes. I've been using some for making mRNA by in vitro transcription and started running into RNAse issues. I'm trying to clean everything up, including the pipettes. I'm not sure these old pipettes can be autoclaved, at least not in one piece, I think I can take them apart and autoclave the parts that might touch the solutions. Is there some way to wash the pipettes out effectively and remove any RNAses?

I use boxes of RNAse free barrier tips, do all my work on new pieces of aluminum foil, use commercial RNAse free water, gloves, labcoat, facemask, etc. However yields from the in vitro transcription kit have been reduced, and yields from the tailing kit are even worse, sometimes losing 90% of the RNA. There's only so much I can do, our building is old and full of dust and the damn pipes above my work bench leak ( all the pipes leak, had 2 major pipe bursts in the last 6 months ).

I'll probably have to get new kits. Just have to prove to my advisor that the kits are contaminated.

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I'd disassemble the pipettes and put the appropriate parts in DEPC solution. I would also consider other issues aside from RNAses, reduced yield can have many different causes. –  Mad Scientist Jul 14 at 20:07
I took apart the pipettes and sonicated the lower parts in RNAse Away, rinsed in ddH2O, then 70% ethanol followed by autoclave. Reassembled with new seals and o-rings and checked the calibration. They seem ok so far. Been using this same kit for about 3-4 months, so it's had a lot of freeze thaws. Will have to replace soon anyway. Running denaturing gels shows smears instead of bands, suggesting degradation. –  user137 Jul 15 at 14:11
I'd also switch out all ingredients for your in vitro transcription if you have any RNAse problem. You might also want to aliquot the stocks further, to limit the damage contamination causes, and to protect the more sensitive stocks. Some stuff just gets bad quicker, DTT should be reasonably fresh, and NTPs also get noticeably worse if they're older. But we also don't use kits but prepare all the stuff in our lab. –  Mad Scientist Jul 15 at 14:16
wipe the pipette's neck with RNAseZap/RNAseAway and use filter tips for your experiment. Degradation is not essentially because of contamination by pipette- check your plasticware- tubes, tips, gel tanks etc. Also check the buffer that you use for your gels (was it autoclaved. is the pH fine). And if your building is dusty, you can perform the experiments in a laminar hood. –  WYSIWYG Jul 18 at 13:01

1 Answer 1

up vote 2 down vote accepted

According to the Gilson Pipettman User Guide you can autoclave the following parts: Tip ejector, Tip holder and the connecting nut (have a look into the PDF linked above if you are not sure about the parts). All other parts can not be autoclaved. Gilson says the following conditions can be used (page 17 in the user guide): Autoclave for 20 minutes at 121°C, 0.1 MPa.

However, when you really have a DNAse contamination, you will not get rid of it with autoclaving, since RNAses are too stable. You can either try the various solutions on the market which made for killing RNAses or simply get a new set of pipettes used only for RNA work (on a different bench in a different lab with a completely different and fresh set of chemicals and plastic ware). And I would recommend filter tips for this work. They are more expensive but in the long run it will save money since you don't have to repeat experiments.

Regarding your leaking pipettes: The pipettes contain rubber rings and teflon seals in the inside which have to be replaced from time to time. The image below shows how (parts C and D), the ring can be purchased from Gilson. If you don't know how to do this work (and have no technician who does) you can also send in the pipettes for service. This costs a bit of money, but is ways better than to use unreliable, leaky pipettes.

enter image description here

And don't worry, Gilson pipettes are pretty good. I have used now more or less all brands on the market and I still prefer them. They only need some service from time to time.

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The pipettes aren't leaking, the pipes are leaking. It's a plumbing problem. I'm cleaning the pipettes right now and I have replacement seals and o-rings, so hopefully this problem will be solved. Can't fix the pipes without a plumber. –  user137 Jul 14 at 20:08
Pipes. I somehow made this into pipettes here. Probably I had too much leaky ones as well :-) –  Chris Jul 14 at 20:11

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