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?