How fast does Taq Polymerase work
Tip 54: Nightmare "Empty PCR Gel" - Ten Little Killers:
There are things that are annoying worse than constant itching. For magazine editors, for example, they are promised articles that arrive three days after the editorial deadline (!) Or sloppy texts that must first be edited in self-tearing detail and then shortened by a third. Scientists have completely different concerns. They like to get upset about graduate students who spill the work area with 32P, and the good TA gets out of her skin when the Postdok has powdered the scales with yeast extract for the eighth time before afternoon coffee.
There is something else that is really annoying, and that is PCR gels that stay empty like opposing football goals on the evening of a busy day. A former colleague usually said laconically in such cases: "Yes, you don't see anything in the cup ...". Not, but you can rule out a few potential sources of error by repeating the experiment. We found ten things "that can kill your PCR" at www.biowire.com. Below are the first four:
- Too many dNTPs, or degraded dNTPs? The former can inhibit your PCR, the latter can make Taq polymerase out of work. The optimal working concentration of nucleotides is between 40 and 200 µM. dNTPs are sensitive to repeated freeze-thaw cycles, so you should aliquot the stock solution and start a new batch frequently (and destroy the old one, otherwise your colleagues will go mad instead of you).
- Forgot to mix the MgCl2 solution? MgCl2 solutions tend to develop a concentration gradient when frozen and should therefore be vortexed (after thawing) before use.
- Wrong MgCl2 concentration? A PCR is an enzyme reaction and therefore logically has an optimal MgCl2 concentration range, normally between 1 and 4 µM. Mg2 + ions form complexes with the nucleotides (dNTPs) and at the same time act as a cofactor for the polymerase used. Determining the optimal Mg concentration is laborious empiricism.
- Inhibitors in the trial? Is it actually clear to you which confused paths your valuable DNA template took before you overturned the PCR master mix? Do phenol, chloroform, EDTA, ionic detergents (SDS, Sarkosyl), xylene cyanol, bromophenol blue or ethanol still hang between the bases and the phosphate backbone? This inhibits the enzyme reaction and the template lies in the cup like a dead dog; exponential increase: Nothing. In this case, give yourself a jolt and run your solution through a cleaning column again - it's quick and does not cost much.
- Bad quality of the overlaying mineral oil? Experienced hobby cooks know that not all oil is created equal. While Witzigmann and Co. have recently started to prefer pumpkin seed pressings, the PCR experimenter (if he does not already have a thermal cycler with a heated lid) should pay attention to nuclease-free oil distillations. Heating (in an autoclave, for example) or exposure to UV rays is also a problem: this often creates reaction-inhibiting hydrocarbon compounds. So don't treat your PCR oil any differently than you treat your high-quality salad oil.
- Too much polymerase used? Then the dreaded "smear" threatens the analysis gel: Unspecifically synthesized pieces of DNA of different sizes run exactly where you suspect the fragment you are looking for and mercilessly cover it up. Many seem stubborn to use 0.5 or 1.0 µl Taq from their stock solution - but usually far less is enough for a good result.
- Wrong primer concentration? Primer, primer, it's always up to the damned primers. If too little is used, there is no (or insufficiently little) reaction product. If you put too much into the approach, things dimerize and again don't do their real job enough: DNA amplification. Concentrations between 0.1 and 1.0 µM should be ideal. Exceptions prove the rule.
- Confused Cycler Program? Sounds banal, but it is not, and not only relevant to paranoid people. It doesn't have to be malicious if "40 Cycles at 94/55/72 ° C" suddenly became "25 Cycles at 94/60/72 ° C". Membrane keyboards are tricky: an unconscious slip, a worn flat button, and you are already amplifying your marginal DNA traces with the program that the bench neighbor used yesterday for his Pfunds isolation ...
- Too much / too little template? Sometimes less is more, especially in PCR. Because excess template catches the primer prematurely during the initial cycles. Too little template, on the other hand, produces only weakly or not at all detectable DNA traces on the analysis gel. Experience has shown that 104 template copies are sufficient for 25 to 30 cycles.
- Poor primer design? Witchcraft is nothing against it: primer design requires a lot of experience. Obvious mistakes such as self-complementarity or extremely long primers (> 30 bp) should of course be avoided. In case of doubt, it is always faster to have new, similar primers synthesized than to laboriously fiddling around with the reaction conditions.
Last changes: 09/08/2004
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