Download 8 cloning - UNM Biology

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
electrophoresis
direct sequencing
Sequence ID (BLAST)
editing
Phylogenetics
GenBank
submission
Qiagen plasmid extraction
Restriction digests
M13 sequencing
Primer design, walking
RT-PCR
gel
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
28-Sep
2-Oct
Restriction enzymes
Southern blotting
Cloning
Gabriela Perales
Carlos Garcia
Timothy McBride
6-Oct
The first sequenced gene
Conrad Greaves
13-Oct
16-Oct
(q)PCR, specificity and
sensitivity
ESTs
20-Oct
BLAST and database searches
Ryan Heimroth
23-Oct
26-Oct
Microarrays
Forensics
Bianca Myers
Jennifer Gutierrez
30-Oct
Genome sequencing , the
$1000 genome
Ayesha Arefin
G
2-Nov
Next generation sequencing
Leslie Janet Lopez
G
6-Nov
9-Nov
13-Nov
16-Nov
Bioinformatics
Epigenetics
non-coding RNA
C-value paradox
Amalia Parra
Clyde Moya
Helen Nordquist
Kelsey Cook
G
20-Nov
Phylogenetic genomics
Jennifer Cooksey
23-Nov
Genes associated with Type 1
diabetes
Katie Kesler
G
G
Krystal Charly
Ian Keller
G
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