Clone libraries PCR: „ Medlin primers“, 51/53°C, 4‘ ext., PE TAQ, Eppendorff cycler; Bias? Cloning: Purify product on crystal violet gel, TOPO XL kit,

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Presentation transcript:

Clone libraries PCR: „ Medlin primers“, 51/53°C, 4‘ ext., PE TAQ, Eppendorff cycler; Bias? Cloning: Purify product on crystal violet gel, TOPO XL kit, > 500 clones Screening: minipreps, 7-enzyme digest, HA gels (~ 200 mp‘s necessary) Sequencing: Quiagen, 528F primer (~ 80% efficiency) Sequence analysis: align (CLUSTAL), construct tree (treecon), sort out duplicates, BLAST, sort out wrong seq., redo tree, determine redundancy (seq. analysis and mp‘s)

Bias of full-length 18S PCR 18S PCRTotal DNA (528f/926R PCR, SSCP gel)

Clone libraries PCR: Medlin primers, 51/53° C, 4‘ ext. PE TAQ, Eppendorff cycler); Bias? Cloning: Purify products on crystal violet gel, TOPO XL kit, > 500 clones Screening: minipreps, 7-enzyme digest, HA gels (~ 200 mp‘s necessary) Sequencing: Quiagen, 528F primer (~ 80% efficiency) Sequence analysis: align (CLUSTAL), construct tree (treecon), sort out duplicates, BLAST, sort out wrong seq., redo tree, determine redundancy (seq. analysis and mp‘s)

Dino. (49) Ciliates (14) Bolido. (6) Chryso. (12) Prasino. (4) Choanofl. Crypto. Chlorarachnio. Traustochytrium-like (7)

PICODIV year 1

Some numbers: Clones to be screened to identify ~ 100 „unique“ ones: ca. 200 Efficiency of Quiagen sequencing: ca. 80% Unique sequences among these: ca „Wrong“ sequences among these: ca. 5 Not clearly identifiable among these: ca. 5 remain: ca unique classifiable sequences/library

Composition of Helgoland clone libraries (individuums) 1 - He He He He001206

Composition of Helgoland clone libraries (species) 1 - He He He He001206

Dinoph. (4/4) Prasinoph. (4/4) Bolidoph. (4/4) Ciliates (3/4) Chrysoph. (3/4) Cryptoph. (3/4) Occurence of classes (pa) „Trausto.-like“ (3/4) Prymnesioph. (2/4) Chlorarachnio. (2/4) Dictyochioph. (1/4) Chloroph. (1/4) Rhodoph. ? (1/4)

No of speciesNo of individuums He00 clones (04/08/10/12)

Occurence of clones throughout the year ~ 11 sequences present in 2 libraries 2 sequences present in 3 libraries NO sequence present in >3 libraries (5 libraries/210 seq. in the alignment)

„normal size“ 18S PCR prod. „small size“ 18S PCR prod. „Normal“ sequencing results Clones could not be sequenced with 528F. Therefore universal primers were used: all were archaebacterial! Unexpected results PCR using eucaryotic, i.e. Medlin primers:

Summary/eucaryotic picoplankton Picoplankton consists of many different classes A significant number of picoplankton species is heterotrophic There are sequences that may belong to new classes There is a high variability among samples Distribution of classes within samples from different sites is very similar Perhaps the most important picoplankton class is the Dinophyta

PICODIV year 2

Single cloneTotal DNAs SSCP (Single strand conformation polymorphism) Do PCR with one phophorylated and one dephosphorylated primer. Digest phosphorylated strand and separate single strands on non-denaturing SDS gel.