26 Jan 2009

Dear user of

's EXCEL tool !

We hope that this small tool which was established with EXCEL 2003, will be of help to you in your daily routine by making your life in the lab a little bit easier. This tool is not only suited to users of Biometra Thermocyclers but is also available to all others as well who work in the field of molecular biology. Find your local distributor of Biometra products at www.biometra.com. General notes: Although this tool has been tested rigorously, we realize that unforeseen errors cannot always be excluded. Therefore, the usage of this tool is offered free of charge at ones own risk. All settings should be entered with care and we welcome any feedback from users. Cells in which parameters have to be entered are marked in light yellow (this color should not be seen on b&w printouts). For your security all other cells are protected. This means that formulas of these cells cannot be deleted nor changed by accident. For example: Given the activation of two cells by entering a sign - like x - will result in a conflict among certain parameters, their color will change from x into x . In such a case one setting has to be removed. The "PCR protocol designer" will be of help in establishing new PCR protocols very quickly. Just define the parameters and activate the features you are interested in - and a protocol will be suggested to you immediately. For example when the protocol contains a temperature gradient and a Biometra Thermocycler from the TProfessional family is used, the gradient temperatures obtained are automatically shown in the "PCR mastermix designer". In this tab you'll find mentioned an example. For using your specific parameters replace the existing parameters by your own ones, please. The sheets "PCR protocol designer" and "PCR mastermix designer" can be printed out if required. Some space is left in order to paste a printout of the gel picture with the results. Printouts of both tabs, the "PCR protocol designer" and the "PCR Mastermix designer" could be stored in the lab journal. In this tab you'll find mentioned an example. For using your specific parameters replace the existing parameters by your own ones, please. Ideally, a blank mastercopy of the protocol designer is stored and all settings are recorded as a new file - why not assign this detail with the relevant information such as "YYYYMMDD_protocol's_name.xls" (esp. suited for chronologic sorting) ?
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Explanations to the protocol settings in tab "PCR protocol designer" Gradient function Different formulas exist for calculating the annealing temperatures of primers. When applying a gradient in the first PCR experiment, the optimal effective annealing temperature can easily be obtained in a single run. All gradient models within Biometra's TProfessional family feature on-screen optimization that is made very easy via: the incremental linear gradient.

Please note that in this EXCEL tool an increment setting of either 1 C or 2 C can be selected. With this capability most temperature ranges are covered that are normally investigated in practice. The increment setting on the unit is flexible. The incremental linear gradient can also be used for amplifying targets, differing in their annealing temperature, simultaneously - given the structure of the protocols with regards to the steps, hold times and number of cycles is identical. Furthermore, with the flexible temperature setting of the heated lid (starting from 30 C), other incubations like digestions, reverse transcriptions, etc. can be conducted, too, without the risk to damage the enzymes by the lid's heat. Again, by using the incremental linear gradient feature different incubations can be performed simultaneously. In the situation the Thermocycler under consideration doesn't feature an incremental linear gradient, the gradient span used can be entered instead. Note with regards to protocol shortening (Fast PCR) Nowadays optimized chemistry is available for performing robust Fast PCRs in order to shorten total run times. Since the hold times advised are extremely short especially for denaturing (TD) and annealing (TA) temperatures, Biometra recommends adjusting the protocols with regards to the cycle conditions (see further background information below). When considering a regular protocol with hold times of 20 sec as the minimum for both steps, denaturation at 94 C
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and an effective annealing at 60 C, the following adjustments with regards to

the temperature settings are essential in order to achieve these temperatures in the samples:

Effective TD Adjusting TD to shortened hold times Effective TA Adjusting TA to shortened hold times

Hold time >= 20 sec 15 sec 10 sec 5 sec 1 sec >= 20 sec 15 sec 10 sec 5 sec 1 sec

@ @ @ @ @ @ @ @ @ @

Set temp 94.0 C 96.0 C 96.5 C 97.5 C 99.0 C 60.0 C 59.0 C 58.5 C 57.5 C 56.0 C

Hold time >= 20 sec 15 sec 10 sec 5 sec 1 sec >= 20 sec 15 sec 10 sec 5 sec 1 sec

@ @ @ @ @ @ @ @ @ @

Set temp 94.0 C 95.5 C 96.0 C 96.5 C 97.5 C 60.0 C 59.5 C 59.2 C 58.8 C 58.5 C

Note: with these protocol adaptions targets of different product lengths have been amplified without further optimization by using a commercial Fast PCR kit. The plastic ware used had an average wall thickness of 0.3 mm and belongs to the class thin walled plastic ware, therefore. Further background information Total run times of PCRs strongly depend on different parameters like ramping rates of the Thermocycler, hold times per step, plastic ware used, and the chemistry used, respectively. The most effective way to significantly shorten the total run time is to reduce the hold times to a minimum. It has been demonstrated that even with conventional Taq polymerase certain targets can be amplified successfully at extremely short hold times (MAI et al., 1998; MARKOULATOS et al., 2003). Since modern Thermocyclers possess considerably higher ramping rates than those units mentioned in these articles, the delay of heat transfer from the wall of the thermoblock's well through the plastic ware and into the sample has to be taken into account: The thermal conductivity of polypropylene is approx. five-fold lower when compared to water. Versus aluminium and silver this factor comes even at hundred-fold (info is available online at 'The Engineering Tool Box'). The thickness of the plastic ware plays an important role as well. According to FOURIER's law the thermal conductivity is reciprocal to the thickness of the material considered. The more simplified explanation:if the wall thickness of the plastic ware is doubled, the heat transfer is halved. It has been shown that for a thin-walled plastic ware having an average wall thickness of 0.3 mm, the delay of heat transfer into the sample takes 20 seconds
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(ELENITOBA-JOHNSON et al., 2008). There is neither a standardization with

regards to the wall thickness nor to the composition of plastic ware. Mimicking the sample temperature in the cyclers control can lead to temperature overshoots and undershoots in the PCR reaction as shown by (KIM et al., 2008). When taking into consideration that the effective window for the biochemical reaction can be very short (MAI et al., 1998; MARKOULATOS et al., 2003), hidden temperature overshooting and undershooting might result in masked optimal conditions which differ from the temperature programmed in the protocol. Taking account of all these factors Biometra's goal is to provide a platform which is open to any kind of plasticware and suited to the more efficient transfer protocols amongst different Thermocyclers. Therefore, algorithms leading to masked temperature overshoots or undershoots have deliberately not been implemented in the cycler control.

Touch down function At a time when a gradient feature has not been available, DON et al., (1991) has developed this type of PCR in order to reduce artefacts in the amplification. This is especially useful if both primers show different effective annealing temperatures - here a touch down PCR can be of help. The maximum effect could be obtained by combining the touch down with the gradient feature. Long-range PCR function Although polymerase molecules are quite heat-stable, many of these deplete during the PCR process. When longer fragments have to be amplified, the yield in particular can be negatively affected by this depletion. In order to compensate for this, in long-range PCR protocols the extension times are slightly increased per cycle from approx. the middle of the protocol. Thus the remaining polymerase molecules compensate for this loss. Lowered deamination function (for small fragments) For short PCR products it has been demonstrated that at lower denaturing temperatures PCR yields can be increased significantly (YAP & McGEE, 1991). Please note that the temperature homogeneity of a Thermocycler is of highest importance when using this feature. Units with a poor temperature homogeneity may fail to denature dsDNA molecules at certain positions in the block. With the introduction of the Biometra TProfessional generation, a new benchmark has been set with regards to the temperature homogeneity.

Note: the entire tab can be printed out for ones own lab journal
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Explanations of the protocol settings in tab "PCR mastermix designer" In most cases, the same PCR reaction is run simultaneously with several different samples. Besides the sample's template DNA, all PCR components are set up in a so-called mastermix. The varying number of samples per PCR run and the differing parameters amongst PCRs make mastermix calculations one of the most unpopular jobs in daily routine. Furthermore, errors in manual mastermix calculations can be one of the main causes of failed PCR experiments. With the "PCR mastermix designer" this work will become easier (although parameters still have to be entered carefully here as well!). In this tab there is already some data mentioned as an example. These can be deleted and replaced by ones own parameters specific for the defined experiment. Given that the gradient feature from the "PCR protocol designer" is activated for gradient enabled units from Biometra's TProfessional family, the gradient obtained is displayed in the "PCR mastermix designer" in the results section. Note: the entire page can be printed out for ones own lab journal and a gel picture of the result obtained after electrophoresis can be pasted at the bottom of the printout.

Explanations of the protocol settings in tab "PCR multiplex designer" In this tab mastermixes for up to decaplex PCR reactions can be designed. By entering the number of targets to be amplified simultaneously into the corresponding cell (from 1 up to 10), the primer pairs to be considered will be marked by "x" in the column preceding the primers. When clicking onto the filter symbol and selecting the marked cells ("x"), only the primer pairs to be used in the experiment will be displayed. This will serve for a convenient clarification of the entire setup. Note: this function is available from EXCEL 2003 only.

Explanations to the settings for tab "Primer calc" In this tab complementary reverse sequences can optionally be generated. Furthermore, the GC content and melting temperature of primers can also be calculated. Results will be obtained for the entire IUPAC nucleotide code. For a primer sequence with ambiguous nucleotides, the lowest and highest
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values will be displayed, therefore.

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The melting temperature is calculated according to MARMUR & DOTY (1962). Although this model is quite basic, it can give results similar to a complex Nearest Neighbor' model (see PANJKOVICH & MELO (2005)). However, one particular outcome of this study was that depending on the calculation method used the estimated melting temperatures show large and significant differences. Therefore, we recommend the use of the gradient feature for determining optimal annealing temperatures for each individual PCR experiment. Furthermore, a tool is provided for calculating the primer concentration. For this, the extinction coefficients from CANTOR et al. (1970) were taken. Last but not least with another tool the optical density (OD) measured can be converted into light absorption measured as a per centage thus giving a feeling for the reliability of the OD obtained.

References:
CANTOR et al., 1970. Biopolymers 9(9): 1059-77 DON et al., 1991. Nucleic Acids Res 19(14): 4008 ELENITOBA-JOHNSON et al., 2008. Biotechniques 44(4): 487-8, 490, 492 IUPAC nucleotide code. URL : http://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/ MolBioReview/iupac_nt_abbreviations.html KIM et al., 2008. Biotechniques 44(4): 495-6, 498, 500 MAI et al., 1998. Biotechniques 25(2): 208-10. MARKOULATOS et al., 2003. J Clin Lab Anal 17(4): 108-12 MARMUR & DOTY, 1962. J Mol Biol 5: 109-18 PANJKOVICH & MELO, 2005. Bioinformatics 21(6): 711-22 The Engineering Tool Box. URL: http://www.engineeringtoolbox.com/ thermal-conductivity-d_429.html YAP & McGEE, 1991. Nucleic Acids Res 19(7): 1713

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01/30/2012

Protocol:

enter name

Target specific PCR parameters

Annealing temperature of primers Length of PCR product (bp) PCR cycles

PCR protocol settings

Please mark the Thermocycler line used TProfessional / TProfessional Standard line TProfessional Basic line Other

Gradient

Suggested PCR Protocol

=cycles

Steps 1 2 3 4

m:s

goto

loops

T(C)

t(s)

(C/s)

Family
featuring the

5 6 7 8 9 10 11 12

incremental linear gradient programming

Important note: usage on own risk only - errors cannot be excluded!

01/30/2012

Protocol:

enter name
your source for Biometra

PCR mastermix setup

No. of reactions: Reactionvolume per rxn: Pipetting error: Reagent Additive 1 Additive 2 Buffer Mg conc of buffer MgCl2/MgSO4 dNTPs (total) Forward primer Reverse primer Polymerase Template ddH2O Stock conc. 5 20 5 rxn l % Final conc. in reaction
products in the UK.
You're located outside of Germany? Find your local distributor at www.biometra.com.

Mastermix setup (l) Additive 1 Additive 2 Buffer MgCl2/MgSO4 dNTPs (total) Forward primer Reverse primer Polymerase ddH2O TOTAL MASTERMIX

10 X 15 mM 15 mM 10 mM each 10 M 10 M 2.0 U/l 200 ng/l

1.0 X 1.5 mM 0.8 mM each 0.3 M 0.3 M 1.0 U 100 ng

10.50 l 0.00 l 8.40 l 3.15 l 3.15 l 2.63 l 74.55 l 105.00 l

Aliquot per single rxn 19.5 l of Mastermix per tube and add 0.5 l of Template

PCR Result
row 1 left 0.0 2 0.0 3 0.0 4 0.0 Temperatures obtained 5 6 7 8 0.0 0.0 0.0 0.0 9 0.0 10 0.0 11 0.0 12 row 0.0 right

Space for pasting the printout of the gel picture

Important note: usage on own risk only - errors cannot be excluded!

01/30/2012

Protocol: enter name

-plex PCR (enter the numbers of targets to be amplified simultaneously, please)

PCR mastermix setup

your source for Biometra

No. of reactions: Reactionvolume per rxn: Pipetting error: Reagent Additive 1 Additive 2 Buffer Mg conc of buffer MgCl2/MgSO4 dNTPs (total) Forward primer 1 Reverse primer 1 Forward primer 2 Reverse primer 2 Forward primer 3 Reverse primer 3 Forward primer 4 Reverse primer 4 Forward primer 5 Reverse primer 5 Forward primer 6 Reverse primer 6 Forward primer 7 Reverse primer 7 Forward primer 8 Reverse primer 8 Forward primer 9 Reverse primer 9 Forward primer 10 Reverse primer 10 Polymerase Template ddH2O Stock conc.

rxn l % Final conc. in reaction

products in the UK.

You're located outside of Germany? Find your local distributor at www.biometra.com.

Mastermix setup (l) Additive 1 Additive 2 Buffer MgCl2/MgSO4 dNTPs (total) Forward primer 1 Reverse primer 1 Forward primer 2 Reverse primer 2 Forward primer 3 Reverse primer 3 Forward primer 4 Reverse primer 4 Forward primer 5 Reverse primer 5 Forward primer 6 Reverse primer 6 Forward primer 7 Reverse primer 7 Forward primer 8 Reverse primer 8 Forward primer 9 Reverse primer 9 Forward primer 10 Reverse primer 10 Polymerase ddH2O

X mM mM mM each M M M M M M M M M M M M M M M M M M M M 2.0 U/l 200 ng/l

X mM mM each M M M M M M M M M M M M M M M M M M M M 1.0 U 100 ng

Aliquot per single rxn -0.5 l of Mastermix per tube and add 0.5 l of Template

Primer: enter name

Enter sequence: (max. 33 bases without gaps) Sequence with gaps is: 5'5'You're located outside of Germany? Find your local distributor at www.biometra.com.

### - 3' - 3' ### ### ### ### ###

Primer characteristics
Length: GC content: mers Melting temperature according to MAMUR & DOTY (1962) TM =

Molecular weight:

Primer dilution
Parameters for lyophilized primer from supplier's data sheet pmol OR nM

Photometrical double-check of primer stock concentration (for DNA primers) Parameters: OD260 measured = l of primer stock PLUS l of ddH2O

(Tip: the average of three measuremens from the same cuvette will reduce errors)

Resulting primer stock concentration = Primer working solution to be prepared: l of 10 M (10 pmoles/l) ###

### low ### 1 cm cuvette path length (note: regularly 1 cm for standard cuvettes) 15,400 L/mol cm - extinction coefficient for chromophore "A" (standard: 15,400) 7,400 L/mol cm - extinction coefficient for chromophore "C" (standard: 7,400) 11,500 L/mol cm - extinction coefficient for chromophore "G" (standard: 11,500) 8,700 L/mol cm - extinction coefficient for chromophore "T" (standard: 8,700) 0.10 1.00 lower OD260 limit (see note below) upper OD260 limit (see note below) ### ### ### ###

Conc.