1. 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

2. All spin at same time

3. 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

4. FIELDTRIP to Sevilleta LTER, Sample collection: Sunday 13 September
FIELDTRIP to Sevilleta LTER, Sample collection:
Sunday 13 September

5. 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

6. Consider putative PCR resultsMW
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

7. 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.)

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

9. BECAUSE OF TEMPLATE INDEPENDENT 3’ A-ADDITION TO AMPLICON

10. 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

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

12. Combine in a snap cap

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

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

15. Plasmid Design I
Selective markers

16. Plasmid Design II
MCS
Visual screening

17. Plasmid Design
BioTech
No ligase

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

19. Alpha complementation, BLUE/WHITE screening
(alpha peptide)

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