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DNA ligation calculation - please help!!

Can someone please help me with this reaction?

Reaction 1 – is to check the transformation works
Volume required to have 50 ng of pBS:_________________
(NB Pipetting less than 1 ul is not accurate, so think about doing a dilution of your pBS)
Volume of T4 ligase buffer: 7.5 μl
Volume of T4 ligase: 0 ul
Volume of PCR product: 0 μl
Volume of water (total reaction must be 15 μl):_______________

I need to dilute some pBS plasmid (because the volume was less than 1ul).
The original concentration is 340ng/ul and the amount is 50ng.
what is the volume of pBS when its diluted, and how do I do the dilution?

I've completely forgotten how to do dilutions, and the total volume has to add up to 15ul (using water to make up the difference)

Can anyone help preferably ASAP??

Reply 1

macpatgh-Sheldon

Dilution factor required = 340/50 = 6.8 i.e, you want a solution 6.8 times weaker.

But some of it is going to be the original solution.

SO add 5.8 ul water to 1 ul of pBS sol-n.

Total vol now = 6.8ul, so you have diluted it by factor of 6.8.

i.e. the new concentration is 340/6.8 = 50ng/ul (which is your requirement)

But you need total volume of 15 ul so take 15/6.8 X 5.8 of water = 12.8ul water AND

Add 15/6.8 X 1 ul of pBS sol-n = 2.2 ul sol-n

Hope this helps,
sorry only saw this Q today!

(edited 7 years ago)