Today Extension product purification (direct sequencing) House Keeping Brief intro TA cloning USE ORIGINAL PROTOCOL for cloning (topo-isomerase, plating) Lecture (TA-)cloning during 1h incubation

All spin at same time

PARASITES AND SNAIL BIOLOGY DNA “identity, possibilities” phylogenetics RNA “intentions” transcriptomics CTAB Trizol gel electrophoresis nanodrop spec Bioanalyzer DNA-free, PCR rDNA/mito TA cloning, B/W screening RT-PCR gel electrophoresis Qiagen plasmid extraction Restriction digests direct sequencing M13 sequencing Sequence ID (BLAST) editing Primer design, walking Phylogenetics GenBank submission 3

FIELDTRIP to Sevilleta LTER, Sample collection: Sunday 13 September http://sev.lternet.edu/about FIELDTRIP to Sevilleta LTER, Sample collection: Sunday 13 September

15 minute powerpoint topics(G+) date topic name 21-Sep Discovery of DNA structure Janette Mendoza 25-Sep Restriction enzymes Gabriela Perales 28-Sep Southern blotting Carlos Garcia G 2-Oct Cloning Timothy McBride 6-Oct The first sequenced gene Conrad Greaves 13-Oct (q)PCR, specificity and sensitivity Krystal Charly 16-Oct ESTs Ian Keller 20-Oct BLAST and database searches Ryan Heimroth 23-Oct Microarrays Bianca Myers 26-Oct Forensics Jennifer Gutierrez 30-Oct Genome sequencing , the $1000 genome Ayesha Arefin 2-Nov Next generation sequencing Leslie Janet Lopez 6-Nov Bioinformatics Amalia Parra 9-Nov Epigenetics Clyde Moya 13-Nov non-coding RNA Helen Nordquist 16-Nov C-value paradox Kelsey Cook 20-Nov Phylogenetic genomics Jennifer Cooksey 23-Nov Genes associated with Type 1 diabetes Katie Kesler

Consider putative PCR results MW PCR reactions yield double or weak bands of “large size” (>800 bp) This will challenge direct and complete analysis by sequencing. Cloning can help! MW

A plasmid is a small DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell. Most commonly found as small circular, double-stranded DNA molecules in bacteria, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids carry genes that may benefit survival of the organism (e.g. antibiotic resistance), and can frequently be transmitted from one bacterium to another (even of another species) via horizontal gene transfer. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. Plasmid: Small circular DNA molecule that replicates independently of the genome. Modified plasmids are used extensively as plasmid vectors for DNA cloning. Figure 8-30The insertion of a DNA fragment into a bacterial plasmid with the enzyme DNA ligase The plasmid is cut open with a restriction endonuclease (in this case one that produces cohesive ends) and is mixed with the DNA fragment to be cloned (which has been prepared with the same restriction nuclease), DNA ligase, and ATP. The cohesive ends base-pair, and DNA ligase seals the nicks in the DNA backbone, producing a complete recombinant DNA molecule. (Micrographs courtesy of Huntington Potter and David Dressler.)

WHY clone? Produce reliable source (amount) of template for stepwise completion of sequencing Separate mixed amplicons Provide universal sequencing primers (Expression of proteins, etc..)

BECAUSE OF TEMPLATE INDEPENDENT 3’ A-ADDITION TO AMPLICON

Clone Duplicate reactions 2x 18S parasite 4 (PCR 3 from group6) Groups 6 and (8+9) 2x 28S parasite 4 (PCR 4 from group6) Groups (1+2) and (5+7) 2x 28S parasite 2 (PCR 4 from group10) Groups 3 and 10 Total 6 cloning reactions, need 12 LB, Kan, Xgal culture plates Label plates with group number, amplified gene, source organism, volume plated. eg: 3,28S,P2,10 or (8+9),18S,P4,100

Manual is online: http://biology.unm.edu/cmadema/4546/TOPOTA.pdf

Combine in a snap cap

1) Do 10 minutes 7) Spread 10 on one plate and 100 on another

Important terms: High copy number plasmid Cloning vector Insert Competent cells Transformation Selection Screen

Plasmid Design I Selective markers

Plasmid Design II MCS Visual screening

Plasmid Design BioTech No ligase

Cloning Modify plasmid Transform bacteria (no previous antibiotic resistance, galactosidase activity) Selection for presence of Plasmid and Insert

Alpha complementation, BLUE/WHITE screening (alpha peptide)

Important terms: High copy number plasmid Cloning vector Insert Competent cells Transformation Selection Screen