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New to molecular and have learned to design primers from google/youtube so any info would help

Would someone be willing to share their protocol for degenerate primer design?

Breakdown: Trying to design degenerate primers to amplify/clone specific GPCR genes from an unsequenced genome of a particular fish.

I'm using Ensembl and Genbank for sequences of closely related fish but the complete gene has only been sequenced in 2 other fish species. I use ClustalOmega for alignment. Is it okay to just use 2 other species for generating degenerates?

1) are there any other good databases for gene sequences?

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If the gene is supposed to be well conserved then you can use the sequence from the related species.

For protein coding genes target the ISH probe to the coding region which is more likely to be conserved.

What you can also do subsequently is to:

  • amplify the CDS
  • do 3' and 5' RACE
  • Clone in a plasmid (do a blunt end or TA cloning)
  • Sequence the insert
  • Design exact primers

are there any other good databases for gene sequences?


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