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Natural Science Forum / Biology / Microbiology / June 2005some numbers.....and primers
First off let me explain that I am putting together a PCR lab of an undergrad microbiology class. I am kinda doing this "on the run" and don't have a lot of time to do incremental experimentation to figure out the best mix and configuration of buffers / primers / etc. So I am doing a whole lot of math work to hedge my bet to insure that the lab works thr first time. Not too big of a task, right? Anyway, doing some calculations on primers in a PCR run I come up with the following.... 1. Purchasing 25 nMole of primers.. 2. Using 20 pMole in reaction Is that enought? Too much? So let's do some math to see what the possible product would be Out comes the calculator, and.... hum... 1. 20 pMole = 120E+11 primered segments .. max 2. 30 PCR cycles = (120E+11)^30 --- Calculator blows up here ---- Doing it by hand .... Equals 2.37E+392 segments at end of PCR run. All inside a little, tiny PCR tube! No! Do not calculate the concentration! STOP! WOW! Now that's a Really Big Number... Now how do you explain this to a room full of undergrads? That's, what? 3.7E+382 times the total number of people on earth? 2.92E+14 times the distance to tau Ceti? Would you even try? > 1. Purchasing 25 nMole of primers.. > 2. Using 20 pMole in reaction [quoted text clipped - 12 lines] > > WOW! Now that's a Really Big Number... Really bigger than what you would get too! Because primers are used during the reaction (they are a part of the newly sythesized fragment) In fact what happens is that you increase the quantity of you template, and synthesize at the maximum 120E+11 fragments. If you ever used all the primers. The tube is much less crowded than you thought! In fact if you admit that you have only one molecule of template at the begiining (that's really not much though) and if you have a 100% functionning PCR, you would end up with only 2E+30, or 1E+9 fragments in the end and that's enough to be detected, and you would still have plenty of primers. And that's normal, because they need to be in excess event at the end if you want your PCR output to be as close as possible of 100% per round (although it will more likely be around 90/95%, last time I cared making the calculation...) Good luck  SignaturePatrick Duriez >First off let me explain that I am putting together a PCR lab of an >undergrad microbiology class. I am kinda doing this "on the run" and [quoted text clipped - 16 lines] > 1. 20 pMole = 120E+11 primered segments .. max > 2. 30 PCR cycles = (120E+11)^30 no. The number of primers is the max possible number of products. Primers are not catalytic. bob >>First off let me explain that I am putting together a PCR lab of an >>undergrad microbiology class. I am kinda doing this "on the run" and [quoted text clipped - 23 lines] > > bob Ahhh yes ... thanks bob. So we are back to the original question. The protocols that I have rounded up call for anything from .1 uMole to 20 pMole. Since the primers are so cheap, should I aim a bit higher than 20 pMole? > So we are back to the original question. > > The protocols that I have rounded up call for anything from .1 uMole to > 20 pMole. Since the primers are so cheap, should I aim a bit higher than > 20 pMole? Coals to Newcastle, Jan. Adding more primers to the excess that is already there simply adds cost without added benefit. SignatureLarry D. Farrell, Ph.D. Professor of Microbiology Idaho State University >> So we are back to the original question. >> [quoted text clipped - 4 lines] > Coals to Newcastle, Jan. Adding more primers to the excess that is > already there simply adds cost without added benefit. I'm with Larry on this one. I've used concentrations close to the 20 pmol for primers and usually gotten fat bands One thing I reccomend is if you are having trouble, try making serial dilutions of the template for each reaction to find out what the optimal template concentration is. This is often faster than prepping a template, then determining the concentration, then diluting to what you think will work. For example from plasmid minipreps, I would try no dilution, 1/10, and 1/100 dilutions. This approach has solved 90% of my problems. The rest have usually been non-specific priming, or primer-dimer problems. >>> So we are back to the original question. >>> [quoted text clipped - 21 lines] > This approach has solved 90% of my problems. The rest have usually been > non-specific priming, or primer-dimer problems. Thanks folks ... If you ever find that you need to build a mesocosm to study the phytoremediation qualities of sub-aquatic vegetation feel free to drop me a line .. now THATS something I know something about! >>>First off let me explain that I am putting together a PCR lab of an >>>undergrad microbiology class. I am kinda doing this "on the run" and [quoted text clipped - 31 lines] >20 pMole. Since the primers are so cheap, should I aim a bit higher than >20 pMole? I am concerned about you trying to do a lab like this without proper pre-running -- esp since you seem unfamiliar with it. But sometimes that happens. Why not involve the students. Maybe have a few do some pre-running for you. Or have the class explore some variables. Half the class uses X and half uses Y. Or each person does 2 conditions, and ... I think you said you were going to use 30 rounds. That is a lot! Unless you need that many, to amplify something very rare, it increases the chance of making a mess. Again, this is something that can be explored -- and such exploration can be instructive to the students. (Sorry, if I have forgotten some of the info you may have posted.) bob > I am concerned about you trying to do a lab like this without proper > pre-running -- esp since you seem unfamiliar with it. Familiar with the theory .. unfamiliar with the practical. I have done this, but all the solutions were pre-mixed and I did not note all of the concentrations used. The protocols that I have read have called for everything from 10 uMole to 20 pMole, hence the question. I intend to do a full end-to-end run prior to turning the students loose, I am just try to hedge my bets toward sucess as I don't have a lot of time to re-do. One of my Engineering profs once said something about a background in physics makes rocket building just slightly easier than having experience. > But sometimes that happens. Why not involve the students. Maybe have a > few do some pre-running for you. Or have the class explore some [quoted text clipped - 7 lines] > students. (Sorry, if I have forgotten some of the info you may have > posted.) Most of the protocols I have seen call for 25 - 35 cycles, so 30 seemed right. I have used 30 cycles before and got nice tight bands and was using bacterial DNA in abundance. I am always open for suggestions. > bob |
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