Week 4:

1
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

2
Restriction Digest of Chromosomal DNA

• Restriction digestion of Chromosomal DNA with
  creates thousands of different fragments (P.
  fischeris genome is 4.2 Mbase)
• Using correct restriction enzyme will leave the
  entire lux operon intact while cutting the rest
  of the genome up

3
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?)

4
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)

5
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)

6
Wednesday checklist

1. Turn in Ex 4 and 6 write up
2. Quantify DNA with spec if not done already
3. Additional clean up of chromosomal DNA
4. Digest the DNA according to the table on p.66
5. Note use XbaI instead of Sal I
6. Note plasmid concentration is not what the book
   says!
7. Prepare a gel while the digestion is incubating
   (use 80 ml as the book says) cover gel with
   buffer and leave until tomorrow

7
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)

8
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

9
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

10
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?

11
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.

12
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

13
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 1-5