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Need help making trypsin edta solution

InfraMan

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Not sure where else to put this.

I'm a med student working in a lab at school. I made a solution following my instructor's directions, but it didn't work. Can someone check the calculations?

Trying to produce 1 liter of .25% Trypsin .53 milli-molar EDTA in DMEM.

Start with 50mM EDTA, 2.5% 10x Trypsin, and plenty of DMEM.

Calculations:

X * 50mM = .53 mM *1000mL
Solve for X to get

X=530/50 = 10.6mL of EDTA

2.5% *Z = .25% * 1000mL

Z = .25/2.5 * 1000 = 100mL Trypsin

100 + 10.6 = 111.6

1000 - 111.6 = 888.4mL DMEM

Thanks in advance!
 
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InfraMan

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Your math works out. That seems like a lot of trypsin. Why do you need so much?

Thanks for checking.

I don't know why he wants me to use so much. I'm still learning at this point. The solution is supposed to be used to lift smooth muscle cells off the surface of a flask where they've been growing.

To answer circumflex's question - I don't know if he wants me to do this as a learning exercise, or it could be that money is tight, and we already have the ingredients laying around.

Thanks, guys.
 

gbwillner

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make sure the DMEM does not have serum in it as well, as that may damper the reaction a tad.

I suppose the troubleshooting should center around why cells aren't coming off the plate. This is an excellent point.

First things first- make sure you wash your cells with PBS before adding the trypsin. Any FBS will prevent the cells from coming off the plate. This step is also cell-dependent- some cells come off easily, and others excrete an extracellular matrix, meaning they are difficult to remove.

I usually wash the cells thoroughly, then apply <1 ml of trypsin solution to a 10 cm plate. I place the plate back in the incubator for a minute or two, then tap the sides of the plate to loosen up the cells. If it works you can see the cells start moving around without a microscope.

I bet your "exercise" is just monkey work to keep you busy. This is not an expensive reagent.
 
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