overnight. Final elution volumes for the mini-prep kit was 30 or 50 C1L, for low copy and

high copy plasmids, respectively, and 200-250 C1L for the maxi-prep kit. Final

concentrations of 0.5 and 1.0 Clg/CIl plasmid were routinely obtained with the Qiagen mini

and maxi-prep kits.

 Digestions, ligations, and transformations. Restriction endonuclease digestions,

ligations, and transformations were performed according to the recommendations of the

manufacturers (New England Biolabs, Stratagene and Invitrogen). For analytical

purposes, restriction endonuclease digestions were normally prepared in a total volume of

20 CIL, including plasmid DNA, enzyme, buffer, ddH20 and occasionally BSA, and then

incubated for an hour at the temperature specified by the manufacturer. Ligations

required two purified double-stranded DNA fragments, a vector and an insert, of known

length and concentration (ng/C1L). DNA fragments were routinely separated in a 0.8 %

agarose gel by electrophoresis and the appropriate sized fragment was excised and

purified using a Qiagen, Inc. QIAquick Gel Extraction kit. The vector fragment

contained the antibiotic resistance gene and the origin of replication. The insert typically

contained the desired gene or a site specific mutation. Two control reactions and two

ligation reactions were set up. The typical reaction was set up in 20-40 CIL and included

vector, insert, ATP, T4 DNA ligase buffer, T4 DNA ligase and ddH20. The first control

reaction was a control for uncut plasmids, containing no insert and no ligase, and the

second, containing no insert, was a control for the vector' s ability to ligate with itself.

The femptomolar concentration (fmol/CIL) was determined from the known size and

measured concentration. Two ligation reactions were then set up, the first had 1 part

vector to 3 parts insert and the second was 1:10. Typically 5-15 fmol of vector was used.