We are using a mechanical pipetting machine to pipette lysed cells in buffer into a 96 well plate from a 24. The protocol asks for 100 uL of lysis buffer to be used in the 24 well plate, but this is not enough for the machine to work with based on this plate size. Is it possible to use more buffer in the 24 well plate of confluent cells (we would end up using approximately 3x as much), or would we be severely affecting our results?
It depends on what you are planing on analyze and how. Your sample will be 3x diluted and depending on your assay that may be undetected. Besides that, 300uL in a 96 well plate can be too much. Depending on the brand of the plate you risk spiling one well into the other.
Hello Colette,
I agree with andre, and would also like to add that excessive buffer can also affect the integrity of your samples.
Can you not avoid the scenario by using a different machine or my using proportionate increase of solutions?
Warm regards,
Venk
Hi Collete
I agree with Mr. fernandez - it seems what you want to do. Of course you have to remember that your sample will be 3 times diluted. But if 100ul is not enaught for your machine you have only 2 options:
1. to put more lysis buffer (maybe not 300ul, but 150 or 200ul - you have to check what is minimum volume for your machine)
2. to do it manually
Regards
Michal
Hi Collete
If you you increase volume of the other solutions such as wash buffer by three fold, you will not face problem because concentration will not be changed. if you want high concentration, use less elution buffer. if the volume is too much for plate, use collection plate (1.1 ml).
I do always have the choice of doing it manually. I suppose I was looking to find out aside from concentration change, if increasing the lysis buffer would affect the integrity of the cells, which Venk answered for me.
Thank you all!
Collette, 300ul in a 24 well plate will be fine as long as you are prepared for the dilution of your cells and constituents on the side of analyses. Extraction efficiency can be decreased slightly by increasing volume. I would presume that you have a well designed experiment where controls and treatments are present on the same plates and therefore your comparisons will be apples to apples and not necessarily absolute values.
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