Hello, all. In my current lab, when we have completed an mRNA extraction, we record the total volume eluted during the final spin, then use that along with determined concentration to equalize all concentration. The current method is to withdraw the eluted volume in a pipette, and discharge air until only fluid remains. However, this seems extremely arbitrary. Not only can the pipette take up a full volume and also withdraw some air, but even a simple twist of 0.2ul can sometimes discharge the volume of air, while other times it can take 3ul worth of twists to discharge an obviously lesser volume of air from the pipette tip.
I have tried to record the mass of the collection tubes, but they seem to vary in mass enough to change the mRNA concentration during calculations.
I believe that regardless of the method, nearly 50ul, or the total amount of water added, is eluted. However, I can get results all the way down to 40ul. I have used both Qiagen and PureLink mRNA kits and this happens with both.
It is my thought that instead of determining the entire eluted volume, we should just determine the concentration of the eluted mRNA, and then take a known volume from the final elution (i.e. 10ul) and normalize that volume. The issue is that I'm just a grad student, and my lab has been doing this assay for much longer than me, so I would like to see if there are any alternate methods of determining the total volume of eluted mRNA at the end of extraction first, instead of jumping straight to this suggestion.
Thanks for taking the time and having the patience to read my question. I appreciate all your answers.
We had two solutions. One, we got a set of empty tubes, and put 10, 20, 30, 40 50, 60 ul of water in. You just compare the volume of liquid in the tube by eye. Our other approach was to normalize volume, ie make the eluate up to 60 ul. We got some clean nuclease free water ready (same as used for elution), set a pipette to 60 ul. We then sucked up the eluate, whatever volume it was, plus some air into the Gilson tip. We the pushed down teh Gilson plunger to push the air out ... dipped the tip in clean water - let go the plunger. That sucked some water in, and the final volume, water plus eluate was 60 ul.. That way we made the volume of eluate up to 60 ul every time. After that, we did concentration. You say your pipette gives inconsistent results? Without getting too technical - you would need to use a new tip every time - as the tips were inside coated so liquids moved smoothly. Also, Maybe the pipette needs servicing. Gilson pipettes depend for accuracy on very small teflon seals and O-rings. If hese are not 100%, then the pipette piston leaks air, and the volumes are not accurate. And The Teflon seals often wore out. We had to check our pipettes regularly (weighing water eluted) and change the seals. Or the workshop did this for us.
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