{"text_format":"3","workbook":"xls","n_sheets":5,"sheets":["Introduction","PCR protocol designer","PCR mastermix designer","PCR multiplex-mix designer","Primer calc"],"not_rendered":{"images":25,"macros":1,"external_links":1,"defined_names":6,"comments":1},"read":"whole","date_system":"1900","formulas":"cached_only"}

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["June 15th, 2010"]
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[null,null," Dear user of ",{"$nulls":6}," 's EXCEL tool V2.2 !"]
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[null,"We hope that this small tool which was established with EXCEL 2003, will be"]
[null,"of help to you in your daily routine by making your life in the lab a little bit easier. "]
[null,"This tool is not only suited to users of Biometra Thermocyclers but is also "]
[null,"available to all others as well who work in the field of molecular biology."]
[null,"Find your local distributor of Biometra products at www.biometra.com."]
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[null,"General notes:"]
[null,"Although this tool has been tested rigorously, we realize that unforeseen errors"]
[null,"cannot always be excluded. Therefore, the usage of this tool is offered free of"]
[null,"charge at own risk. All settings should be entered with care and we welcome"]
[null,"any feedback from users."]
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[null,"Cells in which parameters have to be entered are marked in light yellow."]
[null,"For your security all other cells are protected. This means that formulas of these"]
[null,"cells cannot be deleted nor changed by accident."]
[]
[null,"For example: Given the activation of two cells by entering a sign - like x - will "]
[null,"result in a conflict among certain parameters, their color will change from"]
[null,"x","   into",null,"x",". In such a case one setting has to be changed or removed."]
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[]
[null,"In some cells of the tabs examplified values are entered already. For using "]
[null,"your specific parameters replace the existing parameters by your own ones,"]
[null,"please."]
[]
[null,"All tabs can be printed out and could be stored in the lab journal. In the tab"]
[null,"\"PCR mastermix designer\" there is also some space is left in order to paste"]
[null,"a printout of gel picture with the results."]
[]
[null,"Tip: when storing a blank mastercopy of the protocol designer, this can be "]
[null,"used as template for new experiments. All settings are recorded as a new,"]
[null,"separate file. For chronoligic sorting the following naming might be suited: "]
[null,"YYYYMMDD_protocol's_name.xls."]
[]
[null,"Explanations to the protocol settings in tab \"PCR protocol designer\""]
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[null,"Gradient function"]
[null,"Different formulas exist for calculating the annealing temperatures of primers. "]
[null,"When applying a gradient in the first PCR experiment, the optimal effective  "]
[null,"annealing temperature can easily be obtained in a single run."]
[null,"All gradient models within Biometra's TProfessional family feature on-screen"]
[null,"optimization that is made very easy via: the incremental linear gradient."]
{"$rows_empty":10}
[null,"Please note that in this EXCEL tool an increment setting of either 1 °C or 2 °C"]
[null,"can be selected. With this capability most temperature ranges are covered that"]
[null,"are normally investigated in practice. The increment setting on the unit is flexible."]
[]
[null,"The incremental linear gradient can also be used for amplifying targets, differing"]
[null,"in their annealing temperature, simultaneously - given the structure of the"]
[null,"protocols with regards to the steps, hold times and number of cycles is"]
[null,"identical. "]
[null,"Furthermore, with the flexible temperature setting of the heated lid (starting from "]
[null,"30 °C), other incubations like digestions, reverse transcriptions, etc. can be"]
[null,"conducted, too, without the risk to damage the enzymes by the lid's heat."]
[null,"Again, by using the incremental linear gradient feature different incubations"]
[null,"can be performed simultaneously."]
[]
[null,"In the situation the Thermocycler under consideration doesn't feature an"]
[null,"incremental linear gradient, the gradient span used can be entered instead."]
[]
[null,"Note with regards to protocol shortening (Fast PCR)"]
[null,"Nowadays optimized chemistry is available for performing robust Fast PCRs in"]
[null,"order to shorten total run times. Regularly, the hold times advised are extremely"]
[null,"short. However, hold times of less than 20 seconds may cause a dilemma"]
[null,"the user is confronted with: usually the plastic ware becomes the unknown"]
[null,"variable now (see below chapter further background information)."]
[]
[null,"In experiments we used tubes with a measured average wall thickness of"]
[null,"0.3 mm (\"thin walled\"). Based on this plastic ware, we recommend"]
[null,"adjusting the protocols having hold times of less than 20 seconds for the "]
[null,"denaturating temperature (TD) step and annealing temperature (TA) step, "]
[null,"respectively, as follows:"]
[]
[]
[]
[]
[{"$nulls":9},"Hold time",null,"Set temp",{"$nulls":6},"Hold time",null,"Set temp"]
[null,"Effective TD",{"$nulls":7},">= 20 sec","@","94.0 °C",{"$nulls":6},">= 20 sec","@","94.0 °C"]
[null,"Adjusting TD\nto shortened\nhold times",{"$nulls":7},"15 sec","@","96.0 °C",{"$nulls":6},"15 sec","@","95.5 °C"]
[{"$nulls":9},"10 sec","@","96.5 °C",{"$nulls":6},"10 sec","@","96.0 °C"]
[{"$nulls":9},"5 sec","@","97.5 °C",{"$nulls":6},"5 sec","@","96.5 °C"]
[{"$nulls":9},"1 sec","@","99.0 °C",{"$nulls":6},"1 sec","@","97.5 °C"]
[null,"Effective TA",{"$nulls":7},">= 20 sec","@","60.0 °C",{"$nulls":6},">= 20 sec","@","60.0 °C"]
[null,"Adjusting TA\nto shortened\nhold times",{"$nulls":7},"15 sec","@","59.0 °C",{"$nulls":6},"15 sec","@","59.5 °C"]
[{"$nulls":9},"10 sec","@","58.5 °C",{"$nulls":6},"10 sec","@","59.2 °C"]
[{"$nulls":9},"5 sec","@","57.5 °C",{"$nulls":6},"5 sec","@","58.8 °C"]
[{"$nulls":9},"1 sec","@","56.0 °C",{"$nulls":6},"1 sec","@","58.5 °C"]
[]
[null,"Example of protocol adaption to Fast PCR:"]
[]
[{"$nulls":8},"94 °C",null,null,"1 min"]
[{"$nulls":8},"94 °C",null,null,"30 sec"]
[{"$nulls":8},"60 °C",null,null,"45 sec",null,"30x"]
[{"$nulls":8},"72 °C",null,null,"60 sec"]
[{"$nulls":8},"72 °C",null,null,"1 min"]
[]
[]
[]
[]
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[{"$nulls":3},"94 °C",null,null,"1 min",{"$nulls":7},"94 °C",null,null,"1 min"]
[{"$nulls":3},"97.5 °C",null,null,"  5 sec",{"$nulls":7},"96.5 °C",null,null,"  5 sec"]
[{"$nulls":3},"58.5 °C",null,null,"10 sec",null,"30x",{"$nulls":5},"59.2 °C",null,null,"10 sec",null,"30x"]
[{"$nulls":3},"72 °C",null,null,"60 sec",{"$nulls":7},"72 °C",null,null,"60 sec"]
[{"$nulls":3},"72 °C",null,null,"1 min",{"$nulls":7},"72 °C",null,null,"1 min"]
[]
[null,null,"Note: appropriate chemistry should be used for Fast PCR; hold time at 72 °C might"]
[{"$nulls":3},"  also be shortened then (see manufacturer's instructions)"]
[]
[null,"Further background information"]
[null,"Total run times of PCRs strongly depend on different parameters like ramping"]
[null,"rates of the Thermocycler, hold times per step, plastic ware used, and the"]
[null,"chemistry used, respectively. The most effective way to significantly shorten"]
[null,"the total run time is to reduce the hold times to a minimum. It has been"]
[null,"demonstrated that even with conventional Taq polymerase certain targets can"]
[null,"be amplified successfully at extremely short hold times (MAI et al., 1998;"]
[null,"MARKOULATOS et al., 2003). Since modern Thermocyclers possess"]
[null,"considerably higher ramping rates than those units mentioned in these"]
[null,"articles, the delay of heat transfer from the wall of the thermoblock's well"]
[null,"through the plastic ware and into the sample has to be taken into account:"]
[null,"The thermal conductivity of polypropylene is approx. five-fold lower when "]
[null,"compared to water. Versus aluminium and silver this factor comes even at"]
[null,"hundred-fold (info is available online at 'The Engineering Tool Box')."]
[null,"The thickness of the plastic ware plays an important role as well. According"]
[null,"to FOURIER's law the thermal conductivity is reciprocal to the thickness of"]
[null,"the material considered. The more simplified explanation: if the wall thickness"]
[null,"of the plastic ware is doubled, the heat transfer is halved."]
[null,"It has been shown that for a thin-walled plastic ware having an average wall"]
[null,"thickness of 0.3 mm, the delay of heat transfer into the sample takes 20 seconds"]
[null,"(ELENITOBA-JOHNSON et al., 2008). And here the dilemma occurs - amongst"]
[null,"manufacturers there is neither a standardization with regards to the wall "]
[null,"thickness nor to the composition of plastic ware. Even amongst batches from"]
[null,"the same manufacturer variations in the wall thickness might occur."]
[null,"Mimicking the sample temperature in the cycler’s control can lead to temperature"]
[null,"overshoots and undershoots in the PCR reaction as shown by (KIM et al., 2008)."]
[null,"When taking into consideration that the effective window for the biochemical"]
[null,"reaction can be very short (MAI et al., 1998; MARKOULATOS et al., 2003),"]
[null,"hidden temperature overshooting and undershooting might result in masked"]
[null,"optimal conditions which differ from the temperature programmed in the"]
[null,"protocol."]
[]
[null,"Taking account of all these factors Biometra's goal is to provide a platform"]
[null,"which is open to any kind of plasticware and suited to the more efficient transfer"]
[null,"protocols amongst different Thermocyclers. Therefore, algorithms leading to"]
[null,"masked temperature overshoots or undershoots have deliberately not been"]
[null,"implemented in the cycler control."]
[]
[]
[null,"Touch down function"]
[null,"At a time when a gradient feature has not been available, DON et al., (1991)"]
[null,"has developed this type of PCR in order to reduce artefacts in the amplifi-"]
[null,"cation. This is especially useful if both primers show different effective annealing"]
[null,"temperatures - here a touch down PCR can be of help. The maximum effect could"]
[null,"be obtained by combining the touch down with the gradient feature."]
[]
[null,"Long-range PCR function"]
[null,"Although polymerase molecules are quite heat-stable, many of these deplete"]
[null,"during the PCR process. When longer fragments have to be amplified, the yield"]
[null,"in particular can be negatively affected by this depletion. In order to compensate"]
[null,"for this, in long-range PCR protocols the extension times are slightly increased "]
[null,"per cycle from approx. the middle of the protocol. Thus the remaining polymerase"]
[null,"molecules compensate for this loss."]
[]
[null,"Lowered deamination function (for small fragments)"]
[null,"For short PCR products it has been demonstrated that at lower denaturing"]
[null,"temperatures PCR yields can be increased significantly (YAP & McGEE, 1991)."]
[null,"Please note that the temperature homogeneity of a Thermocycler is of highest"]
[null,"importance when using this feature. Units with a poor temperature homogeneity"]
[null,"may fail to denature dsDNA molecules at certain positions in the block."]
[null,"With the introduction of the Biometra TProfessional generation, a new"]
[null,"benchmark has been set with regards to the temperature homogeneity."]
[]
[null,"Note: the entire tab can be printed out for ones own lab journal"]
[]
[]
[null,"Explanations of the protocol settings in tab \"PCR mastermix designer\""]
[]
[null,"In most cases, the same PCR reaction is run simultaneously with several different"]
[null,"samples. Besides the sample's template DNA, all PCR components are set up"]
[null,"in a so-called mastermix."]
[null,"The varying number of samples per PCR run and the differing parameters"]
[null,"amongst PCRs make mastermix calculations one of the most unpopular"]
[null,"jobs in daily routine. Furthermore, errors in manual mastermix calculations can"]
[null,"be one of the main causes of failed PCR experiments."]
[null,"With the \"PCR mastermix designer\" this work will become easier (although"]
[null,"parameters still have to be entered carefully here as well!)."]
[]
[null,"In this tab there is already some data mentioned as an example. These can"]
[null,"be deleted and replaced by ones own parameters specific for the defined "]
[null,"experiment."]
[]
[null,"Given that the gradient feature from the \"PCR protocol designer\" is activated"]
[null,"for gradient enabled units from Biometra's TProfessional family, the gradient"]
[null,"obtained is displayed in the \"PCR mastermix designer\" in the results section."]
[]
[null,"Note:",null,"the entire page can be printed out for ones own lab journal and a"]
[{"$nulls":3},"gel picture of the result obtained after electrophoresis can be pasted"]
[{"$nulls":3},"at the bottom of the printout."]
[]
[null,"Explanations of the protocol settings in tab \"PCR multiplex designer\""]
[]
[null,"In this tab mastermixes for up to decaplex PCR reactions can be designed."]
[null,"By entering the number of targets to be amplified simultaneously into the"]
[null,"corresponding cell (from 1 up to 10), the primer pairs to be considered will"]
[null,"be marked by \"x\" in the column preceding the primers. When clicking onto"]
[null,"the filter symbol ",{"$nulls":4},"  and selecting the marked cells (\"x\"), only the primer pairs "]
[null,"to be used in the experiment will be displayed. This will serve for a convenient"]
[null,"clarification of the entire setup."]
[null,"Note: this function is available from Microsoft® Excel 2003 only."]
[]
[]
[null,"Explanations to the settings for tab \"Primer calc\""]
[]
[null,"In this tab complementary reverse sequences can optionally be generated."]
[null,"Furthermore, the GC content and melting temperature of primers can also be "]
[null,"calculated. Results will be obtained for the entire IUPAC nucleotide code."]
[null,"For a primer sequence with ambiguous nucleotides, the lowest and highest "]
[null,"values will be displayed, therefore."]
[]
[null,"The melting temperature is calculated according to MARMUR & DOTY (1962)."]
[null,"Although this model is quite basic, it can give results similar to a complex"]
[null,"Nearest Neighbor' model (see PANJKOVICH & MELO (2005)). However,"]
[null,"one particular outcome of this study was that depending on the calculation "]
[null,"method used the estimated melting temperatures show large and significant"]
[null,"differences. Therefore, we recommend the use of the gradient feature for"]
[null,"determining optimal annealing temperatures for each individual PCR"]
[null,"experiment."]
[]
[null,"Furthermore, a tool is provided for calculating the primer concentration."]
[null,"For this, the extinction coefficients from CANTOR et al. (1970) were taken."]
[null,"Last but not least with another tool the optical density (OD) measured can be"]
[null,"converted into light absorption measured as a per centage thus giving a feeling"]
[null,"for the reliability of the OD obtained."]
[]
[]
[null,"References:"]
[null,"CANTOR et al., 1970. Biopolymers 9(9): 1059-77"]
[null,"DON et al., 1991. Nucleic Acids Res 19(14): 4008"]
[null,"ELENITOBA-JOHNSON et al., 2008. Biotechniques 44(4): 487-8, 490, 492"]
[null,"IUPAC nucleotide code. URL : http://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/"]
[{"$nulls":10},"MolBioReview/iupac_nt_abbreviations.html"]
[null,"KIM et al., 2008. Biotechniques 44(4): 495-6, 498, 500"]
[null,"MAI et al., 1998. Biotechniques 25(2): 208-10."]
[null,"MARKOULATOS et al., 2003. J Clin Lab Anal 17(4): 108-12"]
[null,"MARMUR & DOTY, 1962. J Mol Biol 5: 109-18"]
[null,"PANJKOVICH & MELO, 2005. Bioinformatics 21(6): 711-22"]
[null,"The Engineering Tool Box. URL: http://www.engineeringtoolbox.com/"]
[{"$nulls":10},"thermal-conductivity-d_429.html"]
[null,"YAP & McGEE, 1991. Nucleic Acids Res 19(7): 1713"]

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[null,"     General parameters like polymerase parameters as recommended by manufacturer"]
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[{"$nulls":7},"- your source for Biometra's products in Germany."]
[{"$nulls":7},"    You're located elsewhere? Find your local distributor at www.biometra.com."]
[{"$nulls":7},"    You're located outside of Germany? Find your local distributor at www.biometra.com."]
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[null,"Please mark the Thermocycler line used"]
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[{"$nulls":6},"Primer dilution",""]
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[null,"Parameters for lyophilized primer from supplier's data sheet"]
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[{"$nulls":4},"pmol",{"$nulls":3},""]
[null,null,"OR",{"$nulls":5},"",null,""]
[{"$nulls":4},"nM",{"$nulls":5},""]
[{"$nulls":10},""]
[]
[]
[]
[null,"Photometrical double-check of primer stock concentration (for DNA primers)"]
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[{"$nulls":3},"Parameters:",{"$nulls":5},"µl of primer stock  PLUS",{"$nulls":7},"µl of ddH2O",{"$nulls":5},"",""]
[]
[null,null,"OD260 measured =",{"$nulls":6},"(Tip: the average of three measuremens from the same cuvette will reduce errors)"]
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[{"$nulls":12},"Resulting primer stock concentration =","",null,""]
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[{"$nulls":5},"Primer working solution to be prepared:",{"$nulls":11},"µl of 10 µM (10 pmoles/µl)",{"$nulls":10},{"$error":"#VALUE!"}]
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[{"$nulls":28},0,null,null,0]
[{"$nulls":6},1,null,"cm cuvette path length (note: regularly 1 cm for standard cuvettes)"]
[{"$nulls":28},0,null,null,0]
[{"$nulls":6},{"v":15400,"d":"15,400"},null,"L/mol cm - extinction coefficient for chromophore \"A\" (standard: 15,400)"]
[{"$nulls":28},0,null,null,0]
[{"$nulls":6},{"v":7400,"d":"7,400"},null,"L/mol cm - extinction coefficient for chromophore \"C\" (standard: 7,400)"]
[{"$nulls":28},0,null,null,0]
[{"$nulls":6},{"v":11500,"d":"11,500"},null,"L/mol cm - extinction coefficient for chromophore \"G\" (standard: 11,500)"]
[{"$nulls":28},0,null,null,0]
[{"$nulls":6},{"v":8700,"d":"8,700"},null,"L/mol cm - extinction coefficient for chromophore \"T\" (standard: 8,700)"]
[{"$nulls":27},"Conc.","",null,null,""]
[{"$nulls":6},{"v":0.1,"d":"0.10"},null,"lower OD260 limit (see note below)"]
[]
[{"$nulls":6},{"v":1,"d":"1.00"},null,"upper OD260 limit (see note below)"]
[]
[]
[]
[null,"Note to the upper and lower OD260 limits"]
[null,"In photometric application the absorbency - also called optical density (OD) - is measured. Since"]
[null,"nucleic acids absorb light at 260nm wavelength, the OD260 is measured for calculating concentrations."]
[null,"The less light that passes through the cuvette the higher is the OD260 is and the higher the concentration"]
[null,"of nucleic acids, therefore. Mathematically, the OD260 is the logarithmic quotient of light intensity before"]
[null,"entering the cuvette (\"in\") vs. the light intensity leaving the cuvette (\"out\") - so log(in/out)."]
[]
[{"$nulls":14},"The lower the OD260 value measured the higher"]
[{"$nulls":14},"the impact of external factors like fluctuation in"]
[{"$nulls":14},"the light intensity, fit of the cuvette in the holder, "]
[{"$nulls":14},"etc.. Resulting from the curve characteristics, OD"]
[{"$nulls":14},"measurements above a certain value should also"]
[{"$nulls":14},"be handled with care, too. Here, small errors will"]
[{"$nulls":14},"have a big impact that can be figured out below: "]
[]
[{"$nulls":14},"OD260 measured",{"$nulls":5},"=","",null,"% light abs."]
[{"$nulls":17},""]
[]
[{"$nulls":6},"Primer dilution","of primer   enter name here"]
[]
[null,"Parameters for lyophilized primer from supplier's data sheet"]
[]
[null,{"v":1504,"d":"1,504.0"},null,null,"pmol",{"$nulls":3},"For obtaining a 100 µM stock solution resupend lyophilized primer in"]
[null,null,"OR",{"$nulls":5},15.04,null,"µl TE buffer or ddH2O."]
[{"$nulls":4},"nM",{"$nulls":5},"Use as working solution a 1:10 dilution of this primer stock."]
[]
[]
[{"$nulls":30},0]
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[null,"Sequence with gaps is:",{"$nulls":7},"5'-","",{"$nulls":14},"- 3'",null,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,{"$nulls":3},0,0,0,0,null,{"$error":"#DIV/0!"}]
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[]
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[null,null,"Length:","","mers",{"$nulls":13},"Melting temperature according to","Marmur & Doty (1962)"]
[{"$nulls":31},"Molecular weight:",""]
[{"$nulls":3},"GC content:","",{"$nulls":7},"   TM =",null,""]
[]
[{"$nulls":14},""]
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[{"$nulls":6},"Primer dilution",""]
[]
[null,"Parameters for lyophilized primer from supplier's data sheet"]
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[{"$nulls":4},"pmol",{"$nulls":3},""]
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