1. Plant DNA Barcoding using matK some work on new primer sets Dr. Alan Forrest Prof. Pete Hollingsworth Royal Botanic Garden Edinburgh Damon Little, New York Botanic Garden Aron Fazekas, University of Guelph Gao Lian-Ming, Kunming Institute of Botany Sean Graham, University of British Columbia Mehrdad Hajibabaei, CCDB, University of Guelph Maria Kuzmina, CCDB, University of Guelph Hollingsworth, Graham, Little (2011). "Choosing and using a plant DNA barcode." PLoSONE 6: e19254.
2.
3.
4.
5.
6.
7.
8.
9.
10. Angiosperms: recommended work flow Dilute DNA 1:10 1 st ROUND: all samples PCR matK primers xF+MALPR1 1M betaine, 0.2M trehalose, Platinum Taq Clean successful PCR products Sequence clean PCR products 0.2M trehalose Acquire samples and extract DNA 2 nd ROUND: all PCR and SEQ failures 3F+1R or 472F+1248R 1M betaine, 0.2M trehalose, Platinum Taq PCR and SEQUENCE rbcL Clean successful PCR products Sequence clean PCR products 0.2M trehalose >95% matK sequence success rate ALL poor quality sequences/mononucleotide motifs PCR and sequence matK primers xF+ERIR 1M betaine, 0.2M trehalose, Phusion Taq
23. 2-step protocol = >95% 2-step protocol = >95% 2-step protocol = ca. 80% Polypodiales 1-step protocol = 100% Cyatheales Lycophyte and early-diverging lineage primers require testing 1-step protocol = >80% 3-step protocol = >80% Further primer optimization required 2-step protocol = ca. 90% Further primer optimization required
24.
Editor's Notes
Success with 2 pass, with contamenents treated as fail Success with 2 pass, with contaminents treated as fail + 7 recovered with phusion Success with 2 pass, with contaminents treated missing data + 7 recovered with phusion
If anyone asks about ERIR primer, it is same as MALPR1 and comparable results, but with higher annealing temp better suited to use with Phusion Taq
If anyone asks about ERIR primer, it is same as MALPR1 and comparable results, but with higher annealing temp better suited to use with Phusion Taq