{"id":404,"date":"2011-06-08T22:34:32","date_gmt":"2011-06-08T17:34:32","guid":{"rendered":"https:\/\/wordpress.clarku.edu\/polypeet\/?page_id=404"},"modified":"2017-04-14T00:12:47","modified_gmt":"2017-04-13T19:12:47","slug":"primer-information","status":"publish","type":"page","link":"https:\/\/wordpress.clarku.edu\/polypeet\/datasets\/primer-information\/","title":{"rendered":"Primer Information"},"content":{"rendered":"

RNA Polymerase II Largest Subunit (RPB1)<\/strong><\/h3>\n

<\/strong><\/p>\n

For our studies of Polyporales we are sequencing the region between conserved domains A and C (approx. 1400 bp).<\/p>\n

<\/strong><\/p>\n

PCR Primers<\/strong><\/p>\n\n\n\n\n
RPB1-Af<\/td>\nGAR TGY CCD GGD CAY TTY GG<\/td>\nStiller and Hall, 1997<\/td>\n<\/tr>\n
RPB1-Cr<\/td>\nCCN GCD ATN TCR TTR TCC ATR TA<\/td>\nMatheny et al 2002<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Alternatively the sequencing primer RPB1-Int2.2f (see below) gives very good results as a PCR primer when paired with RPB1-Cr (the product is approx. 400 bp shorter). This can be especially helpful if the PCR with Af and Cr fails or gives additional products and you want to avoid cloning.<\/p>\n

Additional Sequencing Primers<\/strong><\/p>\n\n\n\n\n\n\n
RPB1-Int2f<\/td>\nTTM BTC TRC TCG TTT YGC AC<\/td>\nFroslev et al 2005<\/td>\n<\/tr>\n
RPB1-Int2.1f<\/td>\nGCT GAA CGA GSA GTG C<\/td>\nFroslev et al 2005<\/td>\n<\/tr>\n
RPB1-Int2.2f<\/td>\nCGT TTT CGR TCG CTT GAT<\/td>\nBinder et al 2010<\/td>\n<\/tr>\n
RPB1-Int2.1r<\/td>\nGCA CTS CTC GYT CAG C<\/td>\nFroslev et al 2005<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

All \u201cInt2\u201d (also written as \u201ci2\u201d) primers bind at different points of Intron 2 (between domains A and B).<\/p>\n

Usually you will only need one of the \u201cInt2\u201d forward primers to get the desired fragment.<\/p>\n

Sequencing of the PCR product obtained with RPB1-Af- RPB1-Cr usually involves 4 primers: RPB1-Af, RPB1-Int2.2f, RPB1-Int2.1r, RPB1-Cr.<\/p>\n

The primers RPB1-Int2f and RPB1-Int2.1f can be used as additional primers or alternatives to RPB1-Int2.2f if that primer fails.<\/p>\n

If the PCR was done with RPB1-Int2.2f and RPB1-Cr you can use RPB1-Int2.1f as an additional sequencing primer to get a better overlap with RPB1-Cr.<\/p>\n

This is an overview of the expected fragment length and degree of overlap for the RPB1 sequence of Trametes membranacea<\/em> (Click on image for a larger pic).<\/em><\/p>\n

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\n<\/em><\/p>\n

References<\/strong><\/p>\n

Binder, M.,K.H.Larsson, P.B. Matheny, and D.S.Hibbett. 2010. Amylocorticiales ord. nov. and Jaapiales ord. nov.: Early diverging clades of Agaricomycetidae dominated by corticioid forms. Mycologia, 102: 865-880<\/p>\n

Fr\u00f8slev, T. G., P. B. Matheny, and D. S. Hibbett. 2005. Lower level relationships in the mushroom genus Cortinarius<\/em> (Basidiomycota, Agaricales): a comparison of RPB1, RPB2 and ITS phylogenies. Molecular Phylogenetics and Evolution<\/em> 37: 602-618.<\/p>\n

Matheny, P.B., Liu, Y.J., Ammirati, J.F., and Hall, B.D. 2002. Using RPB1 sequences to improve phylogenetic inference among mushrooms (Inocybe, Agaricales). Am. J. Bot.<\/span> 89: 688-698.<\/p>\n

Stiller, J.W. and Hall, B.D. 1997. The origin of red algae: Implications for plastid evolution. PNAS<\/span> 94: 4520 – 4525.<\/p>\n

RNA Polymerase II Second Largest Subunit (RPB2)<\/strong><\/h3>\n

<\/strong><\/p>\n

For our studies of Polyporales we are sequencing the region between conserved domains 5 and 11 (approx. 2100 bp)<\/p>\n

<\/strong><\/p>\n

Important !! <\/strong>Two separate PCR are neccesary in you want to sequence the whole region between domains 5 and 11. However in many studies only the region between domains 5 and 7 is used. When working with very closely related taxa (e.g. species level taxonomy or closely related genera) the most variable region between domains 6 and 7 (approx. 700 bp) can be sequenced using RPB2-b6F and RPB2-b7.1R as PCR and sequencing primers.<\/p>\n

Check also Brandon Matheny\u2019s overview of RPB2<\/a><\/p>\n

<\/strong><\/p>\n

PCR Primers <\/strong><\/p>\n

<\/strong><\/p>\n

PCR 1. Domains 5 – 7<\/strong><\/p>\n\n\n\n\n
RPB2-f5F<\/td>\nGAY GAY MGW GAT CAY TTY GG<\/td>\nLiu et al. (1999)<\/td>\n<\/tr>\n
RPB2-b7.1R<\/td>\nCCC ATR GCY TGY TTM CCC ATD GC<\/td>\nhttp:\/\/faculty.washington.edu\/benhall\/; Matheny (2005)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

<\/span><\/em><\/p>\n

Alternatives to b7.1R are:<\/span><\/em><\/p>\n\n\n\n\n\n
RPB2-b7R<\/td>\nGAY TGR TTR TGR TCR GGG AAV GG<\/td>\nhttp:\/\/faculty.washington.edu\/benhall\/; Matheny (2005)<\/td>\n<\/tr>\n
RPB2-b7R2<\/td>\nACY TGR TTR TGR TCN GGR AAN GG<\/td>\nMatheny et al 2007<\/td>\n<\/tr>\n
RPB2-a8.0R<\/td>\nTCT CKG AAY TTV AGR TAY TCC AT<\/td>\nBinder et al 2010<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Additional sequencing primers:<\/strong><\/p>\n\n\n\n\n
RPB2-b6F<\/td>\nTGG GGY ATG GTN TGY CCY GC<\/td>\nhttp:\/\/faculty.washington.edu\/benhall\/; Matheny (2005)<\/td>\n<\/tr>\n
RPB2-b6R2<\/td>\nGGR CAN ACC ATN CCC CAR TG<\/td>\nMatheny et al 2007<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Sequencing requires 4 primers in total, typically: RPB2-f5F, RPB2-b6F, RPB2-b6R2 and RPB2-b7.1R<\/p>\n

<\/strong><\/p>\n

PCR 2. Domains 7 – 11<\/strong><\/p>\n\n\n\n\n
RPB2-b6.9F<\/td>\nTGG ACN CAY TGY GAR ATY CAY CC<\/td>\nMatheny et al 2007<\/td>\n<\/tr>\n
RPB2-b11R1<\/td>\nTGG ATY TTG TCR TCC ACC AT<\/td>\nMatheny et al 2007<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Alternatives to b11R1 are:<\/span><\/em><\/p>\n\n\n\n\n
RPB2-b10.9R<\/td>\nGTR AAS GGY GTG GCR TCY CC<\/td>\nhttp:\/\/faculty.washington.edu\/benhall\/<\/td>\n<\/tr>\n
RPB2-g11bR<\/td>\nCAA TCW CGY TCC ATY TCW CC<\/td>\nLiu et al. (1999)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Additional sequencing primers:<\/strong><\/p>\n\n\n\n\n
RPB2-f7cF<\/td>\nATG GGY AAR CAA GCY ATG GG<\/td>\nLiu et al. (1999)<\/td>\n<\/tr>\n
RPB2-b8.2R<\/td>\nCTN CGG AAN AGR CCR CGR TC<\/td>\nMatheny et al 2007<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Sequencing requires 4 primers in total, typically: RPB2-b6.9F, RPB2-f7cF, RPB2-b8.2R and RPB2-b11R1<\/p>\n

This is an overview of the expected fragment length and degree of overlap for the RPB2 sequence (Domains 5 -11) of Trametopsis cervina <\/em>(Click on image for a larger pic).<\/em><\/p>\n

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\n<\/em><\/p>\n

References<\/strong><\/p>\n

Binder, M.,K.H.Larsson, P.B. Matheny, and D.S.Hibbett. 2010. Amylocorticiales ord. nov. and Jaapiales ord. nov.: Early diverging clades of Agaricomycetidae dominated by corticioid forms.Mycologia, 102: 865-880.<\/p>\n

Liu, Y.L., Whelen, S., Hall, B.D., 1999. Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Mol. Biol. Evol. 16, 1799\u20131808.<\/p>\n

Matheny, P.B., 2005. Improving phylogenetic inference of mushrooms with RPB1 and RPB2 nucleotide sequences (Inocybe; Agaricales). Mol. Phylogenet. Evol. 35, 1\u201320.<\/p>\n

Matheny, P. B., Z. Wang, M. Binder, J. M. Curtis, Y. W. Lim, R. H. Nilsson, K. W. Hughes, V. Hofstetter, J. F. Ammirati, C. Schoch, G. E. Langer, D. J. McLaughlin, A. W. Wilson, T. Fr\u00f8slev, Z. W. Ge, R. W. Kerrigan, J. C. Slot, E. C. Vellinga, Z. L. Liang, T. J. Baroni, M. Fischer, K. Hosaka, K. Matsuura, M. T. Seidl, J. Vaura, and D. S. Hibbett. 2007. Contributions of rpb2 and tef1 to the phylogeny of mushrooms and allies (Basidiomycota, Fungi). Molecular Phylogenetics and Evolution 43: 430-451.<\/p>\n

Translation Elongation Factor 1-<\/strong>\u03b1 <\/strong>(TEF1)<\/strong><\/h3>\n

<\/strong><\/p>\n

For our studies of Polyporales we are sequencing approx. 900-1200 bp of the gene<\/p>\n

<\/strong><\/p>\n

PCR Primers
\n<\/strong><\/p>\n\n\n\n\n
EF1-983F<\/td>\nGCY CCY GGH CAY CGT GAY TTY AT<\/td>\nRehner and Buckley (2005)<\/td>\n<\/tr>\n
EF1-2212R<\/td>\nCCR ACR GCR ACR GTY YGT CTC AT<\/td>\nRehner and Buckley (2005)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Alternative to <\/span><\/em>EF1-2212R:<\/em><\/span><\/p>\n\n\n\n
EF1-2218R<\/td>\nATG ACA CCR ACR GCR ACR GTY TG<\/td>\nRehner and Buckley (2005)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

In our experience EF1-2212R<\/strong> works better than EF1-2218R as a PCR and sequencing primer.<\/p>\n

Additional sequencing primers:<\/strong><\/p>\n\n\n\n\n
EF1-1577F<\/td>\nCAR GAY GTB TAC AAG ATY GGT GG<\/td>\nRehner and Buckley (2005)<\/td>\n<\/tr>\n
EF1-1567R<\/td>\nACH GTR CCR ATA CCA CCR ATC TT<\/td>\nRehner and Buckley (2005)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Sequencing requires 4 primers in total, typically: EF1-983F, EF1-1577F, EF1-1567R and EF1-2212R<\/p>\n

This is an overview of the expected fragment length and degree of overlap for the TEF1 sequence of Earliella scabrosa <\/em>(Click on image for a larger pic).<\/em><\/p>\n

\"\"<\/a>
\n<\/em><\/p>\n

References<\/strong><\/p>\n

Matheny, P. B., Z. Wang, M. Binder, J. M. Curtis, Y. W. Lim, R. H. Nilsson, K. W. Hughes, V. Hofstetter, J. F. Ammirati, C. Schoch, G. E. Langer, D. J. McLaughlin, A. W. Wilson, T. Fr\u00f8slev, Z. W. Ge, R. W. Kerrigan, J. C. Slot, E. C. Vellinga, Z. L. Liang, T. J. Baroni, M. Fischer, K. Hosaka, K. Matsuura, M. T. Seidl, J. Vaura, and D. S. Hibbett. 2007. Contributions of rpb2 and tef1 to the phylogeny of mushrooms and allies (Basidiomycota, Fungi). Molecular Phylogenetics and Evolution 43: 430-451.<\/p>\n

Rehner, S.A., Buckley, E., 2005. A Beauveria phylogeny inferred from nuclear ITS and EF1-a sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 97, 84\u201398.<\/p>\n

PCR protocol used for RPB1, RPB2 and TEF1<\/strong><\/h3>\n

<\/strong><\/p>\n

Protocol designed by Zheng Wang<\/a><\/p>\n

1. 94 \u02daC for 2:00<\/p>\n

2. 94 \u02daC for 0:40<\/p>\n

3. 60 \u02daC for 0:40<\/p>\n

minus 1 \u02daC per cycle<\/p>\n

4. 72 \u02daC for 2:00<\/p>\n

5.\u00a0 Go to 2, 8 times<\/p>\n

6. 94 \u02daC for 0:45<\/p>\n

7. 53 \u02daC\u00a0 for 1:30<\/p>\n

8. 72 \u02daC for 2:00<\/p>\n

9. Go to 6, 36 times<\/p>\n

10. 72 \u02daC\u00a0 for 10:00<\/p>\n

11. 15 \u02daC\u00a0 for ever<\/p>\n

12. END<\/p>\n

 <\/p>\n","protected":false},"excerpt":{"rendered":"

RNA Polymerase II Largest Subunit (RPB1) For our studies of Polyporales we are sequencing the region between conserved domains A and C (approx. 1400 bp). PCR Primers RPB1-Af GAR TGY CCD GGD CAY TTY GG Stiller and Hall, 1997 RPB1-Cr … Continue reading →<\/span><\/a><\/p>\n","protected":false},"author":29,"featured_media":0,"parent":88,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"onecolumn-page.php","meta":{"ngg_post_thumbnail":0,"footnotes":""},"class_list":["post-404","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/wordpress.clarku.edu\/polypeet\/wp-json\/wp\/v2\/pages\/404","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/wordpress.clarku.edu\/polypeet\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/wordpress.clarku.edu\/polypeet\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/wordpress.clarku.edu\/polypeet\/wp-json\/wp\/v2\/users\/29"}],"replies":[{"embeddable":true,"href":"https:\/\/wordpress.clarku.edu\/polypeet\/wp-json\/wp\/v2\/comments?post=404"}],"version-history":[{"count":0,"href":"https:\/\/wordpress.clarku.edu\/polypeet\/wp-json\/wp\/v2\/pages\/404\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/wordpress.clarku.edu\/polypeet\/wp-json\/wp\/v2\/pages\/88"}],"wp:attachment":[{"href":"https:\/\/wordpress.clarku.edu\/polypeet\/wp-json\/wp\/v2\/media?parent=404"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}