# So confused!

1. Sep 2, 2004

### sobored

here is a part of the protocol we did:

a)spin down the chloroplast solution and decant the supernatant. add 4ml water to the pellet.

b) add 10ul proteinase K ( concentration= 20mg/ml) to get a final concentration of 50ug/ml.

-> how do they get 10ul? which formula do they use? i don't think that they use the dilution formula (C1 XV1= C2 X V2) since we don't know the total volume of the solution. it can't be 4ml, because when we add proteinase K the volume of the solution will increase.

c) then add slowly 0.25ml 10% or 0.125ml 20% SDS to get a final concentration of 0.5% SDS.

-> again, how do they come to 0.25ml 10% or 0.125ml 20% SDS to get a final concentration of 0.5% SDS?

2. Sep 3, 2004

### chem_tr

Hello

Please remember that some protocols do not reveal some data in order to maintain full usage. Your approach is a logical one, so I conclude that there is something not disclosed.

10 microliters of proteinase K with a concentration of 20 mg/mL makes 10 micrograms of enzyme solution. I am sure you are aware of this. If you calculate the overall concentration, you may find the same result.

With the same way, they probably calculated the overall concentration of SDS in the final solution and required that you provide the same.

Regards
chem_tr

3. Sep 3, 2004

### sobored

thank, you so much for helping me out! but i am still very confused, so i hope you will keep helping me out.

i have found the answer for a)

X/10ul=20ug/ul
X= 200ug

200ug/4ml= 50ug/ml.

this is what they get.

but, i can't figure out the answer of b) since the unit is in percent.

i am so confused so i really hope for replies!
thank you so much again!!!!

4. Sep 3, 2004

### iansmith

Staff Emeritus
The formula for percentage is the same you used for calculating the correct concentration of proteinase K. An easier to see it, the % represent Weight per volume. So you had 10 g of SDS into 100 mL of water so 10%=[1g/10mL]

5. Sep 3, 2004

### Moonbear

Staff Emeritus
Just to add a clarification to iansmith's post: A weight/volume solution expressed as a percentage can also be read to have units of g/100ml. It means exactly the same thing. Using g/100 ml in your calculations makes it far easier to balance your units and make sure your math is correct.