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tiredofscience 07-31-2012 05:00 PM

Determining morpholino concentration
 
Hi all,

I am injecting some fish and seeing a phenotype but I am not sure of the concentration my fish are receiving (ng total). Can you guys help me figure it out?

I diluted my stock to 1mM and inject the fish with 4.6nL of 0.4mM solution. The MW of the MOs are 8474 and 8458

Jon Moulton 08-01-2012 05:15 PM

Re: Determining morpholino concentration
 
0.4 mM = 0.0004 mole/l
4.6 nl = 4.6E-09 l
mole/l * l = mole
1.84E-12 mole


For 8474 g/mole
mole * g/mole = g
1.55922E-08 g = 16 ng


For 8458 g/mole
mole * g/mole = g
1.55627E-08 g = 16 ng

I think most reviewers would call that a high dose of Morpholino, high enough that the observed phenotype might be caused by off-target RNA interactions. Have you confirmed your concentration of Morpholino in the stock solution by UV spec?
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If some of the Morpholino has come out of solution, then you could be delivering a lower dose than you think -- in your case, that would be good as you would be getting a phenotype at a lower dose than you thought (but you need to measure the actual concentration of the solution to ascertain whether or not that is the situation). Be sure to heat the oligos prior to aliquoting, dilution or injecting; you want all of the oligo to be uncomplexed in solution, available as active antisense.

tiredofscience 08-02-2012 05:23 PM

Re: Determining morpholino concentration
 
Thank you! I thought I was going crazy when I was getting numbers that high...

I'll spec them right away!


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