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Replacing one reagent by another in a protocol (Please help)

volume reagent PCR

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5 replies to this topic

#1 madelingirly

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Posted 01 November 2012 - 12:53 AM

Dear Guys,
Tomorrow I would perform my first PCR experiment and I had made this stupid mistake, I ordered incorrect reagentPosted Image .

I will use some kit for amplification of RNA "Takara cell Amp" and I should ordered specific DNA polymerase, conc is 5um/ul, then added dNTP mixture and buffer separately.
However, what I ordered is the same reagent but premixed with dNTP and buffer, the same conc but in different volume.
so if I need around 5.25ul from the original reagents after mixing.
I would use 25 ul from the same premix reagents to get the same conc but volume is higher by 3.25ul
So I guess buffer conc may change as the volume become larger
have any one face similar situation and share it with mePosted Image
Please help

#2 leelee

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Posted 01 November 2012 - 07:38 AM

the same conc but in different volume.
so if I need around 5.25ul from the original reagents after mixing.
I would use 25 ul from the same premix reagents to get the same conc



This doesn't make sense. Either it is the same concentration (in which case the same volume would have the same amount of reagents) OR it has a different concentration so you will need a different volume to get the same amount of reagents.

And what do you mean the volume will be higher by 3.25?

I'm really confused by what you are trying to say, but I'll attempt to answer based on what I think you are saying...

So you usually make up your master mix, and this would require 5.25ul of dNTP, polymerase and buffer. Which you would then top up with water and your template?

But you've bought pre made master mix by mistake, and to get the same amount of dNTP and polymerase, you'd need 25ul of master mix- and then you still need to add your template (and water?).
If this is the case, the buffer is likely 2x instead of say 10x (which is probably is for your normal reagents), so you'll need to add template and water to a total volume of 50ul.


Ok actually no, I can't make sense of what you are asking sorry... could you try explaining again?

#3 phage434

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Posted 01 November 2012 - 11:27 AM

Some commercially prepared master mixes are at around 1.1x, with the idea that they will work with modest additions of primer and template volumes, as long as those are not too large. PCR is pretty insensitive to exact concentrations, so this works out quite well. Saves pipetting water (which also saves contaminating your reaction with your "water").

#4 madelingirly

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Posted 01 November 2012 - 05:52 PM

Dear

leele

Thanks for ur concern.
what I should do :
use 5.25 ul of reagent then added H2O to become 21.25.
Then added my reaction mixture and primers 3.25 ul
so final volume of reaction is 25 ul
So i will skip adding the water, as if I added the water this means my buffer will not be 2X but 0.4X and every thing will be diluted

However, according to what I bought by mistake:
I would use 25 ul of premix reagent, the I have to added 3.25 ul (reaction mixture and primers).
so the final volume would be 28.25 ul
this was my question.
The DNA polymerase and bases and buffer would be used in the same amount as original reagents but conc of course will be different as the volume is changing.
but looking to the bigger picture I need 1.25 unite of polymerase and I got it, but in 28 ul not 25.
wNow lets go back to my inquiries;
I will have extra volume and no water???
so what I should expect
Shimaa

#5 madelingirly

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Posted 01 November 2012 - 05:55 PM

Dear
phage434

Thanks for being optimistic, this is very encouraging.
hopefully, every thing may goes well from this experiment

#6 leelee

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Posted 01 November 2012 - 09:19 PM

Thanks for clearing that up :)

So the pre made master mix is 1x? I only ask because I've never bought a 1x before, only 2x...

Anyway, given the small difference in volume and taking into consideration phage434's comments- I agree it should be fine.

Especially as you haven't yet done the reaction with the 25ul, so its not like you'll be comparing results from one type of set up to another.

Good luck :)





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