• Marking and grouping wells
• Linking treatment to control groups
• Assigning the housekeeping gene(s)
• Calculating the normalized and fold change values

View the tutorial and find out how easy analysis can be!

Achieving a good standard curve fit is important for calculating unknown analyte concentrations (e.g. cytokine or chemokine) using xMAP assays. A good curve fit gives confidence in the final result. However for many the task can be daunting because there are so many variables:

1. Do I treat replicates at individual points or use the average value?

2. What if a replicate fails?

3. Do I use 4PL, 5PL or some other model equation?

4. When do I use a weighting algorithm and which on should I use?

5. How do I know I have achieved the best curve fit?

MasterPlex QT has always offered advanced features for customers who like to manipulate standard curve fitting parameters manually. The latest version of MasterPlex makes it even easier to quickly ascertain the quality of your fit and tweak it on the fly by selecting different model equations, weighting algorithms or throwing out saturated and/or failed standard replicates. The new interface puts everything you need to optimize your curve fit in to one tab so you can quickly make changes and compare results instantly.
The new Best Fit feature takes curve fitting to the next level. Of course you still have the option to tweak your curve fit to heart’s content but who has the time!

Test drive the new Best Fit feature now by downloading the fully-functional trial demo of MasterPlex QT v4.0!

We are now happy to introduce the new Term Licensing options for our MasterPlex QT and GT Luminex data analysis software.  Term Licensing durations include the 3, 4, 5, 6, 7, 8, 12, or 24 month options.  The software license expires at the end of the purchased term and also includes all major upgrades released during the term.

For those that do not need to run analyses 24/7, this may be the cost-efficient choice for you.  To find out more details, please visit the MasterPlex QT product page or the MasterPlex GT product page at the MiraiBio Store.

The following procedure guides you through utilizing BLAST against NCBI’s sequence databases.

1. Launch DNASIS MAX and import the Tutorial1.fsa file which is located in the
C:\HSK_DB\TutorialData directory.

Fig1. After loading the tutorial data.

2. Click the DNA – Compare tab and select the Internet – Blast Search option.

Fig2. DNA – Compare tag.

3. The Analysis dialog will appear.

Fig3. Analysis Dialog – In the Analysis Dialog, you will find the Parameter and Execute buttons.
The Parameter button is used for changing the parameters of an analysis.

4. Press the Parameter button and a dialog box will appear.
NCBI BLAST is set as the default URL.

Fig4. Internet Blast Search Parameterset dialog box.

5. Press the Setting… button to edit the parameters of NCBI BLAST.

Fig5.　NCBI Advanced BLAST Search dialog.

6. Once the configurations have been set, press the OK button.
To discard any changes, press the Cancel button.

7. Press the Execute button to send the query.
The time it takes to retrieve your results will depend on the size of the sequence, the database, and your internet service provider.  Once your results come back, it will be displayed like in Fig6.

Fig6. A sample result of the BLAST search in DNASIS MAX.

1. Launch DNASIS MAX and import the Tutorial1.fsa file which is located in the
C:\HSK_DB\TutorialData directory.

Fig1. After loading the tutorial data.

2. Click the DNA - Compare tab and select the Blast Search option.

Fig2. DNA – Compare tag.

3. The Analysis dialog will appear.

Fig3. Analysis Dialog – In the Analysis Dialog, you will find the Parameter and Execute buttons.
The Parameter button is for changing the parameters of an analysis.

4. Press the Parameter button and a dialog box will appear.

Fig4. BlastParameters dialog box.

5. Select the Blast program type from the Program name box.

• blastp – compares an amino acid query sequence against a protein sequence database
• blastn – compares a nucleotide query sequence against a nucleotide sequence database
• blastx – compares a nucleotide query sequence translated in all reading frames against a protein sequence database
• tblastn – compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames
• tblastx – compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

6. Set the E-value or Expect value in the Expectation value field. This value represents the number of times you would expect to see such a match (or better) merely by chance. The closer the value is to zero, the less likely the event is. To re-iterate, the smaller the E-value, the more significant the match is and the lower number of hits you’ll see.  The default value is 10.

7. Select the intended database from the Nucleotide Database.
Select the In-house-NA for this tutorial.

8. Once the configurations have been set, press the OK button.
To discard any changes, press the Cancel button.

9. Press the Execute button to start the blast analysis.

Fig5. A sample result of the Blast search in DNASIS MAX.

For more information on DNASIS MAX, please visit the DNASIS MAX product page or download the FREE trial.

1. Launch DNASIS MAX and import the Tutorial1.fsa file which is located in the

C:\HSK_DB\TutorialData directory.

Fig1. After loading the tutorial data.

2. Click the DNA – Search tab and select the Primer Design option.

Fig2. DNA – Search tab.

3. The Analysis dialog will appear.

Fig3. Analysis Dialog – In the Analysis Dialog, there are the Parameter and Execute buttons.  The Parameter button is for changing the parameters of an analysis.

4. Press the Parameter button and a dialog box will appear.

Fig4. PrimerParameterEditor dialog box.

5. Once the configurations have been set, press the OK button.
To discard any changes, press the Cancel button.

6. Press the Execute button to start the primer design.

Fig5. A sample result of primer design in DNASIS MAX.

For more information on DNASIS MAX, please visit the DNASIS MAX product page or download the FREE trial.

To import these templates into MasterPlex QT:

1. Import a plate file and complete your analysis
2. Click on the Report Generator icon
3. Select Custom Report for Report Type and click Generate
4. Click on the View button from the Custom Report window
5. Click on the Import button from the Style Sheet Manager window
6. Browse to the template file that you have downloaded and press Open

To use a template:

1. Import a plate file and complete your analysis
2. Click on the Report Generator icon
3. Select Custom Report for Report Type and click Generate
4. Click on the View button from the Custom Report window
5. Select the template to use from the Style Sheet Manager and press Apply

Depending on the type of data the template outputs, you might see an output that is difficult to view or make sense of. There are generally 2 types of output files:

1. CSV (comma separated value) – This file can be easily detected since all the values are separated by commas. To view this file in Excel, press the Save button and save the file with a .csv extension. If you have Excel installed, it will automatically recognize this file type and parse it correctly when you open it.
2. XML – The output from this template type will contain many tags such as <Workbook…> or <Cell…>. Press the Save button and save the file with a .xml extension. Next locate the file and right-click on it. Select the Open With option and choose Excel. This will launch Excel and you should be able to view your data in the workbook.

For more information on MasterPlex QT, please visit the MasterPlex QT overview page or download the FREE trial.

What can SmartNote offer to you?

• SmartNote has many analysis tools including popular tools such as Blast, ClustalW, Primer3 and much much more. You can run a single analysis on one sequence or save time by running a batch analysis on multiple sequences. If you don’t see a tool that you would like, then just request it and we will try our best to add it as soon as we can.
• You can easily share sequences, results, pages, and even your entire notebook with colleagues.
• Store and organize your sequences using our tagging system to find them easily later on.
• Track articles for your sequences. Do you find yourself constantly looking for new articles related to the sequences you are working on? Now, you can sit back and relax and let SmartNote automatically do it for you.

We are only skimming the surface here. If you are interested in the full potential of SmartNote and what it can do for you, then check it out for yourself!

1. Design your PCR primers to be 18-30 oligo nucleotides in length. The longer end of this range allows higher specificity and gives you space to add restriction enzyme sites to the primer end for cloning.

2. Make sure the melting temperature (Tm) of the primers used are not more than 5°C different from each other. You can calculate Tm with this formula: Tm = 4(G + C) + 2(A + T)°C

3. Aim for a Tm between 65 and 70°C for each primer over the region of hybridization

4. Use an annealing temperature (Ta) of 10 to 15°C lower than the Tm.

5. The GC content of each primer should be in the range of 40-60% for optimum PCR efficiency.

6. Try to have uniform distribution of G and C nucleotides, as clusters of G’s or C’s can cause non-specific priming.

7. Avoid long runs of the same nucleotide.

8. Check that primers are not self-complementary or complementary to the other primer in the reaction mixture, as this will encourage formation of hairpins and primer dimers and will compete with the template for the use of primer and reagent.

9. If you can, make the 3′ end terminate in C or A, as the 3′ is the end which extends and neither the C or A nucleotide wobbles. This will increases the specificity.

10. You can avoid mispriming by making the 3′ end slightly AT rich.

11. Use the right software. OK, so it’s 11 tips. Using the right software is a great way to automate these steps and minimize errors, especially when you have to design primers for many sequences. DNASIS SmartNote includes several primer design tools and is also a lab notebook that automatically keeps a record of your analysis results. You should definitely give it a try. Click here to sign up a free account.

If you prefer traditional desktop software, take a look at DNASIS Max instead.

References:

http://rothlab.ucdavis.edu/protocols/PrimerDesign.html

http://www.biochem.ucl.ac.uk/bsm/nmr/protocols/protocols/oligo.html

http://www.protocol-online.org/prot/Molecular_Biology/PCR/PCR_Primer/

http://www.mcb.uct.ac.za//pcroptim.htm

1. You can now export individual results (summaries or raw) from your lab notebook as PDF files or print them out.

2. The PrimerX tool has been added to support primer design for mutagenesis. It even works with multiple sequences!

3. You can now extract email addresses from your GMail or Yahoo mail accounts to make it easier to invite your colleagues to collaborate with you through DNASIS SmartNote.

4. If you find yourself using the same tools frequently, you’ll love this feature: when it’s time to pick a tool for an analysis, you can now see the 5 most recently used tools listed at the very top and just click them to quickly launch the analysis.

Coming soon… an exciting new way to share data with colleagues!

Some users said they needed more help getting started using it. This is not surprising, since DNASIS SmartNote’s design is innovative in two ways – it’s a meta-application (an application that uses other applications) and it’s also the first bioinformatics tool we know of that combines a lab notebook, sequence analysis and social networking.

Others said they want to be able to import sequences in formats other than FASTA. I’m happy to report that GenBank support will soon be available on the live site.

Finally, we are starting to get requests to support specific workflows, such as designing primers for mutagenesis. One user suggested adding a view that shows both DNA and protein sequences. We would love to hear from other DNASIS SmartNote users so we know where to focus our efforts to best meet users’ needs. So please send us feedback or post to this shared blog. Together, we can make DNASIS SmartNote even better!