Download Week4 Lab Lecture

Week 4:
• Today:
– Clean up Chromosomal DNA
– Restriction Digestion of Chromosomal DNA from P.
leiognathi
– Pour Gel for next time
• Thursday:
– Florescence Quantification of DNA
• Note that we will use the gel quantification (Ex. 9A, not 9B),
to quantify the DNA
– Ligation of Digests into Plasmid Vectors
Restriction Digest of Chromosomal
DNA
• Restriction digestion of
Chromosomal DNA with creates
thousands of different fragments
(P. fischeri’s genome is 4.2
Mbase)
• Using correct restriction enzyme
will leave the entire lux operon
intact while cutting the rest of the
genome up
Creation of a library of restriction
fragments (Wednesday)
• The chromosomal digestion can be ligated
into plasmids with compatible sticky ends
• Ideally this creates a library of all of the
restriction fragments from the digest
• These can be transformed in to competent
E. coli and which can then be screened for
the plasmid containing the fragment of
interest. (What is the phenotype of the lux
operon fragment?)
Additional Chromosomal cleanup:
• Phenol will inhibit digestion and ligation of
DNA.
• If you suspect phenol is present in your
chromosomal DNA extract with chloroform
(see handout)
Changes to Ex. 8
• We are using P. leiognathi instead of V fischeri.
Xba I must be used instead of Sal I
• The concentration of plasmid DNA is not
necessarily what the book says
– look at your notes from last Thursday and calculate
the amount you will need to equal 1 ug
• Stop at part B #10 of Ex 8 (leave the gel covered
in buffer for tomorrow)
Wednesday checklist
1.
2.
3.
4.
Turn in Ex 4 and 6 write up
Quantify DNA with spec if not done already
Additional clean up of chromosomal DNA
Digest the DNA according to the table on p.66
1. Note: use XbaI instead of Sal I
2. Note: plasmid concentration is not what the book
says!
5. Prepare a gel while the digestion is incubating
(use 80 ml as the book says) cover gel with
buffer and leave until tomorrow
Thursday
• Gel Florescence Quantification of DNA
and Ligation of Digests and Plasmid
Vectors (Ex. 9A)
• Ligation of digests into plasmid vectors
(Ex. 10 part I)
Agarose Gel Florescence
Benefits of this method over spectroscopy
– Much smaller amounts of DNA can be used
– Et. Br. is more selective to DNA than to RNA
and proteins, so contaminants influence the
measurement less than with spectroscopy
• Drawbacks
– Hard to quantify chromosomal digests
– Some variance in EtBr binding to DNA
Method:
• We will use a standard curve of known
amounts of DNA (Lambda Hind III ladder)
and compare the florescence of this to the
florescence of our DNA
• Same florescence = same amount of DNA
on the plate
Ligation of the restriction fragments
into plasmids (Ex 10 pt. 1)
• Rational:
– Efficient ligation of fragments into plasmids
depends on having the correct ratios of vector
(Plasmid) to insert (chromosomal digest)
About 3 insert to 1 vector works best
– We know the amount of DNA in the digest but
how many fragments are there?
Ligation continued
• Because of this, we will optimize the
ligation with different ratios of amounts
DNA (p. 84)
• Notes on T4 ligase
– Activity inhibited by monovalent cations
– Buffer contains ATP that is required for
ligation, so keep it on ice until use.
Wednesday Checklist
• Quantify vector and insert DNA digests
with agarose gels
• Calculate volumes of DNA needed for
ligations
• Set up ligation reactions and place in
thermocycler at the end of class for
overnight incubation
Due Thurs Feb 8:
• Ex. 5 (Chromosomal DNA extraction) with
questions 1-3
• Ex. 7 (Spectrometry) with questions 1-4
• Ex. 8 (Restriction Digest) with questions 15