1. Today House Keeping (talks, sequencing) Follow-up on cloning
Transformants?
Plasmid extraction
Insert check by restriction digest
(gel....)

2. 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

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. 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

5. 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?

6. Do you have
the desired insert?
How to check
Presence,
Size of insert

8. Gelelectrophoresis Cast gel, load samples, run, take photo……
How long is that going to take?

9. Gelelectrophoresis

10. What to expect? Insert size: Plasmid size: Pattern on gel:
Or digest?
Insert size:
Plasmid size:
Pattern on gel:
What primers to use?

12. Do you have the desired insert? How to check Presence, Size of insert
Plasmid(linear)
insert
EcoRI digest of constructs

13. Sequencher?