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PCR set-up calculation nightmares.

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#1 PhilS



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Posted 27 November 2009 - 03:48 PM

I never look forward to setting up PCR reactions, so I'm trying to make it as easy as possible. The problem comes when I have multiple things to set up, for example, the worst situation would be 4 templates, 3 primer sets, 5 temperatures. I've set these up before, but they take a fair bit of planning to get the right calculations for each mastermix (e.g a mastermix for each template, each primer set, then put these into 5 tubes for each of the 5 temperatures). I normally screw up somewhere, particularly with the excess I calculate for each mastermix, wither a lot over or under. I normally use a spreadsheet but I have to change it each time depending on the number of variables, so I was thinking of setting up a number of spreadsheets e.g.

Spreadsheet configuration: mastermixes
x templates, x
x templates, z primers, x + z
x templates, y temperatures, z primers x + y + z

I generally have math phobia although the math is not hard, but when I started to set this up I wondered if I'm over thinking the problem, and wanted to see what others do. Maybe I should forget doing the optimal multiple mastermixes and just pipette the templates and primers into the final tubes themselves, and then prepare one mastemix and add to each. What do you do, or what would you do in this situation?


#2 Vini



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Posted 28 November 2009 - 05:38 AM

Hi Phil, in such instances, I do make multiple master-mixes which include everything except the template......i add th etemplate separately and then add the mix......but i m sure e'one has a diff way of doing it n really u hv to find out what way it suits u........ :lol: my method maybe confusing to someone else....
How about concentrating really well when setting up the reactions???? :lol: just kidding.... :lol: i guess ur phobia gets better of u........all the best!

#3 Adrian K

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Posted 29 November 2009 - 07:35 PM

I do my PCR each time not less than 50 tubes. I do prepare a master-mixes using spread sheets.

I just share my thought on your problem based on my experience
For your problem: 4 templates, 3 primer sets, 5 temperatures

4x3x5 = 60 variables to be tested.
For extra tubes (to minimized pipette error), I use 15% : 60 x 15% = 9...I rounded up to 10.

Thus prepare master mix 70 tubes. You will never get less by then. When you put into individual tube, you will get around 63 tubes, and probably lost the rest. 3 extra tubes you can use as -ve or +ve control.

To do the PCR, I will prepare:
1) Master mix(taq + mgcl + buffer + DMSO etc. without top up water) in one tube
2) template in another tube(s)
3) and primer mixes (1 sets, 2 sets 3 sets..... with top up water) in another.

So, using a 96-well, put in the master mix first, then add templates. Finally mix well with your primers, seal and run your PCR.

Hope this helps.

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#4 PhilS



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Posted 30 November 2009 - 09:06 PM

Many thanks,
I found a bug in my spreadsheet that was causing the errors, I'd always end up with less and couldn't figure it out before.
cheers for the tips and help,

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