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.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 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 1- 5