I want to make sure if my calculations are correct. I have 370ng/ul stock of my 3'UTR reporter plasmid, I have to use 10ng during the transfection, does that mean that I use 0.03ul of the stock to obtain 10ng?
Secondly, I have a stock concentration of 236ng/ul of pre-miR, I must use 30nmol/l (which is 30nM).
The length of the pre-miR is 106bp, and from an online calculator (which I assume is designed for dsDNA) it determined that 236ng/ul = 3656.5 nM. Is this correct? Then I wanted to work out how much of this stock I must use if I must use 30nM per well (of a 6 well plate). This worked out to 24ul. This seems like a bit much, seeing as I was only supplied with 50ul of stock.
I would appreciate it if someone can assist me :)
hii,
for the first bit of your question. I would diulte down a small volume of 370ng/ul to a lower concentration for instance 5ng/ul or something. The formula you can use to calculate that is = (final concentration/initital concentration) x volume. So in this case the final concentration is 5, initital concentration is 370 and volume lets say is 100uL. Therefore you need to add 1.35uL of stock into 98.6uL of dH20. and then you have a stock of 5ng/uL. You can then add 2 uL of that to obtain 10ng.
hope this helps.
Thanks, yes I thought I would dilute it down if I indeed understood it correctly that I should divide 10 by 370?
Hi Stefanie:
Q1) if you have a 370ng/ul stock - that means there's 370ng of plasmid DNA in each ul. so to pipette 10ng in a reaction you need 0.027ul. personally i think that's a very small amount to attempt to pipette, you may want to dilute down the stock so you're pipetting a larger volume - unless you're going to scale that up in a master mix or something.
Q2) if you have the sequence of the pre-miR (which is available on miRBase) - you can enter it into the following program and it will calculate the FW for you:
http://www.basic.northwestern.edu/biotools/OligoCalc.html#helpMW
you can use a molarity calculator such as:
http://www.graphpad.com/quickcalcs/Molarityform.cfm
to calculate the molar concentration using the 'molarity from mass and volume' calculator.
i think your calculation might be off a bit though...very roughly - using an average extinction coefficient of 308 - for a 106nt oligo at 236ng/ul you're looking at a concentration of about 7uM
good luck with your experiments - hope that helps
Hi Stefanie,
What product are you using for the pre-miRNAs? Are you using miRNA mimics (eg. Qiagen) or miRNA precursors (Ambion/Life Tech)?
Keith
Hi
I am using genecopoeia's precursor miRNAs, so it the hairpin structure cloned into a vector, so I am unsure if I can even calculate the molecular weight in this way?
If you're using a plasmid to express your miRNA - I agree, you could simply titrate the miRNA precursor plasmid (and its equivalent NC miRNA plasmid) against a constant amount of your 3'-UTR reporter plasmid when co-transfecting. Do you have any synthetic miRNA mimic you can also try?
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