PCR optimization - (Aug/10/2005 )
am here again and new to mol bio and i thank everyone who is guiding me and sharing their expertise and experiences. I just want to ask again this time with optimization...
i started pcr run using the protocol in journal and so far it amplified okay ( so far control gene) was amplified. Should i start varying the magnesium concentration? if that is the case, how am i going to appoach or add it in my samples? should i add the volume directly to reaction tube or add it in the master mix?
after magnesium chloride, should i play around with annealing temperature still? or stick with the annealing temperature i programmed? number of cycles is already 45. Should I retain it too?
You can vary the final MgCl2 concentration from 1.0-3mM in 0.5mM increments. You add this into the mastermix (in with your buffer). If you have a 10X buffer that already has MgCl2, it will usually be at a working concentration of 1.5mM (and in that case I'd skip the 1mM). Remember this is final working concentration of MgCl2 in your reaction
The easiest way to vary the annealing temperature is to use a gradient PCR block (ask around - someone may have one you can borrow). This way you can alter the MgCl2 and the annealing temperature in one PCR. (for example, you can check 4 MgCl2 concs and 12 annealing temperatures for two samples in a 96 well PCR gradient block).
If not, then you can start with an annealing temperature of ~5 C below the lowest Tm of the primer pair, or (since it is a published result) just increase the temperature by a few degrees (1-2C) until you get the best result. For each temperature you test, make sure you have a variety of MgCl2 concentrations too - this way you will optimise for both.
45 cycles does seem like alot to me. If you get nice strong bands with your MgCl2 and annealing temperature optimised PCR then you can try reducing it to (say) 40 or 35. I'd leave that until last though!
I dont have a gradient PCR so still struggling to optimize my PCR profile haha
Yeah, I hear you.
My last lab had a spiffy new one that I could do the above optimisation with. My new lab only has a 24-tube capacity. It takes too bloody long!
*sit and twiddle thumbs*
You can simulate varying the annealing temp by using different PCR volumes and relatively short annealing times (say 10sec). The larger the volume the lower the annealing temp. This is due to the thermal mass of reaction.
Longer automated DNA sequencing reads
thank you nicole. i will try that. i think we have a gradient pcr. i will check. usually in optimization, how many samples are ideally needed to be run? 2 tubes in each condition? thanks.
It depends on what you want to amplify. Is it a gene on a conserverd region of human/animal/plant genomic DNA? Then I'd say 1 or 2 tubes on every condition would be fine. If however you want to amplify a gene of a very varied genome (virusses or so) then I'd try several different (and diverse) samples for every condition. I once optimised a PCR on a plasmid and just ran a gradient PCR with just one sample for every condition and was succesful (just ran 10 temperatures and 3 different Mg concentrations).
What does MgCl2 do in the PCR reaction?
There are probably plenty of other posts in this, but I'll just give you a brief description. Mg2+ ions have a three important functions in a PCR reaction.
1) Form a soluble complex with dNTP's which is essential for dNTP incorporation
2) Necessary co-factor for taq polymerase activity
3) Increases the Tm (melting temperature) of primer/template interaction (i.e. it serves to stabilize the duplex interaction )
Note: #1 & #2 is a bit of a balancing act. If dNTP concentration is too high, then Mg2+ will become rapidly depleted and PCR will become inhibited.
If Mg2+ content is too low then the result is little or no PCR product. If it is too high then mispriming can result. The ideal concentration for your reaction is something that you need to play around with. Typical troubleshooting, as mentioned by Nicole, usually entails running a Mg2+ gradient between .5 and 3 mM for normal PCR reactions and sometimes as high as 4-5 mM for more specialized PCR.
For more information you can check out this linkYale
While the replies have been informative, I'm curious about your original question. If your PCR is working, why are you trying to optimize it? What do you expect to get out of an "optimized" reaction that you are not getting out of the one that is working? Don't mess with success, I say.