Also try to use I (Inosine) as a base for positions with 4-fold degeneracy such as A/T/G/C while grouping the nucleotide sequences (end to end) using Clustal W or any such free multiple sequence alignment tool, would allow you to group the sequences and thus "design several such degenerate PCR primers" to use them taxa-wise (say within plants, or across 2 families in animals, or across several families of plants/ animals). A little twitching of the PCR conditions using a few gradient PCR experiments would help you amplify much species-specific gene of interest. So if degenerate forward primers based on conserved residues at N-terminal ends of the proteins and oligo(dT)18 reverse primers that are complimentary to the poly-AA Tails in mRMA, then one can get amplicons of varied lengths for different organisms. The forward degenerate PCR primers needs to be designed manually looking at the aligned nucleotide sequences of the homologous sequences as submitted in NCBI-GenBank or similar public resources.
Universal primer list serial cloner how to#
I guess that with sufficient patience and determination, you might manage to cook down about 20 000 sequences manually, but it would be serious toil.Īnswers are :degenerate PCR primers based on Arabidopsis and Oryza sequences. Serial Cloner Tutorial 17,003 views 54 Dislike Share Rissa B 23 subscribers Very brief tutorial on how to use the Serial Cloner application.
I'm working with some Bioinformaticians at the University of Pune in India to try to automate this process. All libraries prepared with the current Illumina library. The list of most variable regions for use within species has little in common with. For more information, see the bulletin Spiking custom primers into the Illumina sequencing primers. The primers worked quite well, though, which was gratifying. Universal barcoding primers were the least likely to work (65 success). For your assembled sequence, choose the format you prefer by checking the box for a FASTA, GenBank, or Serial Cloner format and click on Export assembled sequence to export. Click on Export oligos to generate a text file of the primers. It took me about three days uninterrupted work and it was a real grind. You have the option of listing your suggested primers in an IDT compatible format or a FASTA format. Doing this, I managed to cook about 500 sequences down to five. I solved the problem in the Borrelia case by manually editing out identical sequences, grouping them into manageable groups and then manually editing the consensus sequence from the alignment (the automatically-generated consensus merely gives you the most frequent nucleotide what you need is ambiguity codes indicating the actual variation in the sequences). Your problem is at least an order of magnitude more difficult you can't align all the flagellin genes in the database - even if it's computationally possible, the alignment would be impossible to read.
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