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Tag Archives: lab notebook
10 Tips For Designing PCR Primers That Work
Since PCR primer design is one of the most widely used features of DNASIS SmartNote, we did some research and put together a list of the top 10 tips for designing PCR primers that work. When designing oligonucleotide primers for PCR, it is helpful to keep some considerations in mind to optimize the output and specificity of your experiment. Here are some tips gathered from experts to get you started:
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/
Track articles related to your sequences and congrats to Boris Zinshteyn!
DNASIS SmartNote now tracks articles related to your sequences as they are published. You can see a list of recent articles relevant to all your sequences by clicking the “Articles” tab at the top, or click the “Related articles” link next to each sequence to see only articles for that sequence. Coming soon – you’ll be able to clip individual articles into your lab notebook!
Congratulations to Boris Zinshteyn for winning the Top Feedback prize of a $25 Amazon.com gift certificate! We still have plenty of gift certificates available, so please take a couple of minutes to tell us what you think about DNASIS SmartNote and especially how we can improve it for you.
Export/print results, new PrimerX tool, invite GMail + Yahoo Mail contacts and more!
New this week:
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!
DNASIS SmartNote – Feedback from Limited Release
DNASIS SmartNote was recently released to a limited group of scientists and attracted some great feedback. Here’s a quick summary:
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!
Welcome to the DNASIS SmartNote Blog!
Collaboration is fundamental to life science research. Publications, conferences and lab meetings have their place, but most scientists would benefit from more granular collaboration with a wider audience. That’s why we’ve added a blog to SmartNote – to foster a community of users who help each other get the most out of SmartNote and discuss ideas that can help their research.
Use this blog to post:
- protocols and get help troubleshooting your experiments
- experiment results from your lab notebook that you want to share and invite others to comment on- relevant events, shows and conferences
- hot topics in life science research- job postings
- ideas for how to improve SmartNote
- anything else of potential interest to the community of SmartNote users
Then, invite your colleagues to read and comment on your posts. The most popular posts will automatically be listed on this page, so you can easily access the information that others find most useful or interesting.So go ahead and get started!