Plasmid isolation, help with dilution step? - (Apr/16/2009 )

Hi all,

I have isolated and spec'd my plasmid DNA and got a concentration of 6.645 ug/ul. My lab likes to aliquot the plasmid DNA as 200 ug of DNA, and I need to dilute the plasmid DNA concentration to 1ug/ul. How do I do it? I have a total volume of 98 ul and with this template we use, it says to use 30.10 ul for 200 ug. But this concentration is still at 6.645 ug/ul?

Thanks. sad.gif

you want to dilute your plasmid to 1 ug/uL.
your initial concentration is 6.645 ug/uL, you have to dilute 6.645 times.
your initial volume is 98 uL. the final volume will be 98*6.645 = 651 uL
so, you add 553 uL to your 98 uL (651 - 98 = 553 uL) to get a final concentration of 1 ug/uL

if you want aliquote of 200 ug, you will have to aliquote 200 uL (1 ug/uL = 200 ug/200 uL)

actin_crazy on Apr 16 2009, 06:25 AM said:

No, you add 30.1ul (200ug) of stock to a new tube, then you add MQ or buffer to 200ul, so you have 1ug/ul of plasmid and it is no longer 6.645ug/ul

The point is the amount of plasmid is fixed at your initial stock, you can adjust the concentration by adding different volume of buffer.

actin_crazy on Apr 16 2009, 03:25 PM said:

To have your DNA at a concentration of 1ug/ul, you need to dilute it 6.645 times (i.e. add 6.645 times the volume you already have).

Other way to calculate this is: C1V1=C2V2
where C1 is your initial concentration (6.645ug/ul), V1 your initial volume (98ul), C2 your final concentration (1ug/ul) and V2 the final volume you need; so in this case:
V2=6.645*98/1= 651.21ul so you need to add 553.21ul of whatever your DNA is diluted in, to your 98ul.

To your other question, 30.1ul of your 6.645ug/ul will contain 200ug (yes the concetration is still 6.645ug/ul as long as you dont add anything else to it, which will dilute it and reduce the concentration, but will still be 200ug of DNA).

Oh! finally, if what you want is 200ug DNA aliquots @ 1ug/ul, you just have to split your 1ug/ul DNA (diluted as explained above) into 200ul aliquots.

hope this helps

almost a doctor on Apr 16 2009, 06:43 AM said:

Thanks for the replies. I tried diluting and aliquoting using the method described above however I spec'd my 200 ul aliquots and got a concentration of less than 1ug/ul?

are your pipettes properly calibrated?

pipettes and spectrophotometers are accurate +/- some error.

mdfenko on Apr 17 2009, 11:44 AM said:

As far as I know. My pipettes are brand new and the spectrophotometer I use is a common one used by everyone on the floor.

are you sure that your tips are seated properly?

how is your pipeting technique?

are you sure that your predilution concentration was correct?

how far from 1 are you?