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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)

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Clone libraries

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  1. 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) Clone libraries

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

  3. 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) Clone libraries

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

  5. PICODIV year 1

  6. Some numbers: • Clones to be screened to identify ~ 100 „unique“ ones: ca. 200 • Efficiency of Quiagen sequencing: ca. 80% • Unique sequences among these: ca. 50-60 • „Wrong“ sequences among these: ca. 5 • Not clearly identifiable among these: ca. 5 • remain: ca. 40 - 50 unique classifiable sequences/library

  7. Composition of Helgoland clone libraries (individuums) 1 - He000427 2 - He000803 3 - He001005 4 - He001206

  8. Composition of Helgoland clone libraries (species) 1 - He000427 2 - He000803 3 - He001005 4 - He001206

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

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

  11. 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)

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

  13. 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 Summary/eucaryotic picoplankton

  14. PICODIV year 2

  15. Total DNAs Single clone 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.

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