Mobility Shift Assay and Cloning Using Lambda attP DNA

PART A: Cloning Using Lambda attP DNA

This portion of the experiment will probably be completed when you come to lab. The attP DNA will be amplified with PCR, using specific primers which flank the attP region. A phenol-chloroform extraction will be performed on the PCR fragment. A phenol-chloroform extraction is a very common technique in Molecular Biology and is used to separate proteins from DNA. We will remove free nucleotides (dNTP's) and the Taq Polymerase. Phenol is an organic compound. The protein will sequester in the organic layer while the DNA remains in the aqueous. The DNA is then precipitated with 95% ethanol and washed with 70% ethanol, similar to the alcohol treatment of the miniprep DNA. The wash will remove the last traces of the dNTP's as well as other salts. The dried DNA is resuspened in TE and will be ready to cut with restriction enzymes. It is important that the Taq Polymerase and extra dNTP's be removed before the DNA is cut. Otherwise, the Taq may continue to synthesize DNA and fill in the "stick ends" left by the restriction enzymes. The phenol-chloroform method is used because Taq cannot be heat inactivated (why?).

The PCR reaction will amplify a 1194 bp fragment of the Lambda attP region, including the three IHF binding sites. Cleavage by restriction enzymes will leave a 593 bp attP fragment for use in cloning and MSA.
 

2. Recombination of attP PCR and Plasmid pBLU

Both the attP PCR product and plasmid pBLU will be cleaved with the enzymes BamHI and BbuI. There are a few new concepts introduced in this cloning experiment:

• use of a compatible buffer for the BamHI-BbuI "double digest".  Molecular biologists use the term double digest when two enzymes are used simultaneously to cut DNA. We have already done several double digests with BamHI and HindIII. However, both those enzymes have the same optimal buffer. Now we will have to determine the best buffer for our BamHI-BbuI digest. Obviously, the two enzymes must also have compatible incubation temperatures.
• using enzymes which are isoschizomers of each other. Isoschizomers are two enzymes that recognize the same restriction site. In our experiment, we will take advantage of the fact that BbuI and SphI are isoschizomers. The insert DNA contains a BbuI site, while the vector contains an SphI site.

3. Transformation of E. coli and Plating onto LB/Amp+X-gal plates

The transformation process will be the same as our previous experiment. Hopefully, we will have frozen competent cells and will not have to make more. Since frozen competent cells can be stored at -80'C for up to three months, tt will be necessary to add a viability control to our other transformation controls.

In this experiment, we will screen for the presence of positive clones by using the "blue-white" test. We will spread X-gal (5-bromo-4-chloro-3-indoyl-D-galactoside) on our LB/Amp plates. A pBLU plasmid with a functional lacZ gene will produce b-galactosidase. Bacteria containing this plasmid will form a blue colony because the enzyme will metabolize the X-gal substrate. If the lacZ gene is disrupted by inserting foreign DNA, then the gene cannot produce b-galactosidase and the colony will be the normal beige.
 

4. Purification and Identificatio of pBatt Recombinants

Overnight cultures of white colonies will be used to isolate plasmid DNA via the miniprep procedure. The DNA will be cleaved with restriction enzymes and analyzed on an agarose gel to determine if we have any successful pBatt recombinants.
 

PART B: Mobility Shift Assay of Lambda attP and IHF Protein