today house keeping (talks, sequencing) follow-up on cloning
DESCRIPTION
Sequencing Many worked, analysis due! Check and analyze using “Chromas”(freeware) and “Sequencher”, Genecodes provides teaching license for use during the semester! Computers available for sequencher, or use your own; genecodes.com/support/sequencher-server-download http://technelysium.com.au/?page_id=49, http://nucleobytes.com/index.php/4peaksTRANSCRIPT
Today
• House Keeping (talks, sequencing)• Follow-up on cloning• Transformants?• Plasmid extraction• Insert check by restriction digest• (gel....)
Sequencing• Many worked, analysis due!• Check and analyze using “Chromas”(freeware) and
“Sequencher”, Genecodes provides teaching license for use during the semester!
• Computers available for sequencher, or use your own; genecodes.com/support/sequencher-server-download http://technelysium.com.au/?page_id=49, http://nucleobytes.com/index.php/4peaks
PARASITES AND SNAIL BIOLOGY
“identity, possibilities”phylogenetics
“intentions”transcriptomics
PCRrDNA/mito
BioanalyzerDNA-free,
direct sequencing
gel electrophoresisnanodrop spec
Sequence ID (BLAST)editing
Phylogenetics
electrophoresisRT-PCR
gel
CTAB
Trizol
TA cloning, B/W screening
M13 sequencing
Primer design, walking
Qiagen plasmid extraction Restriction digests
DNA
RNA
GenBank submission
Work schedule today• Each group handout.• “Purelink Quick plasmid extraction"
for 4 samples (1.5 ml bacterial culture).• EcoRI digest on each sample for insert
check on gel.• Talk• Sequencher
Some questions:
Did you obtain transformants?
What is the composition of plasmids in W and B colonies?
How do you recover plasmids?
What likely happens in the Purelink Quick plasmid extraction protocol?
How can you check for the size of the insert?
Do you havethe desired insert?
How to checkPresence,Size of insert
Gelelectrophoresis
• Cast gel, load samples, run, take photo……
• How long is that going to take?
Gelelectrophoresis
What to expect?
• Insert size:• Plasmid size:• Pattern on gel:
• What primers to use?
Or digest?
http://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/
http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.html
Do you havethe desired insert?
How to checkPresence,Size of insert
Plasmid(
linea
r)ins
ert
1 2 3 4
EcoRI digest of constructs
Sequencher?