I have noticed that my reaction volume has been decreased by 2-3 ul after the restriction digestion of plasmids with different restriction enzymes at 37 °C. I am sure that there is no or very minute pipetting error while preparing the reaction mixture.
Please suggest me some possible reasons and explanations.
Thank you.
Dear Nikhil,
a decrease of 2-3 ul of sample can be due to the normal evaporation of the sample. Even if the eppendorf is closed, a little bit of water can evaporate and thus decrease the volume, especially if the digestion is long. To recover a little bit of volume you can try to spin your samples, at least to throw down the water on the cap and on the walls of the eppendorf. However, a decrease of volume lower than 10% should not have effects on your digestion.
Enrico
Nikhil: Quality tubes do not allow evaporation. However, there may be reflux that can be recovered by centrifugation as mentioned above. Yet, evaporation in small volume reaction volumes occurs due to low laboratory humidity during reaction assembly. The evaporation of 5-10% can alter enzyme specificity. To limit evaporative influences, assemble on ice and/or incresase volume.
A small decrease of 2-3 microlitres of sample volume is generally observed after restriction digestions kept for too long time at 37C due to normal evaporation process. To avoid this, the eppendorf tubes can be sealed with sealers such as parafilm and centrifuged after the digestion process to recover the sample.
It would be worth double checking your reaction volume prior to incubation. While tubes can (and do) lose volume due to evaporation, it is possible that your pipet is off by 10%. If it hasn't been calibrated in the past year or so now might be a good time for a service call. You can check the accuracy yourself by pipeting and weighing water (1 ml is 1 g).
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