Purification of the product of the 2nd PCR reaction, Digestion set up

A small sample of the 2nd PCR reaction will be run on gels, to ensure that the product was acheived. The rest of the PCR will be purified for later steps.

PCR Reaction Procedure

1. Pipet a small 1 microliter dot of DNA loading buffer onto a peice of parafilm
2. Remove 10 microliters from the PCR tube and mix with that dot via pipet
3. Pick up the mixed smample and load it into on fo teh wells on teh gel, using a gel with small wells. A DNA ladder is loaded into the first lane
4. Run the gel at 120V for 1 hour.
5. Stain in ethidium bromide for apporximately 15 minutes and visualize.

As the gel runs, perform the purification.

Purification of 2nd PCR product

1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. Since 40 microliters of sample used, 200 microliters of buffer is used.
2. Place a QIAquick spin column in a provided 2ml collection tube
3. Apply the sample to the QIAquick colum and centrifuge for 1 minute to bind the DNA
4. Discard the flow-through. Place the QIAquick column back into the same tube.
5. Add 0.75 ml Buffer PA to teh QIAquick column and centrifuge for 1 minute, to was the sample.
6. Discard flow thru and place the QIAquick column back into the same tube and centrifuge for an additional minute.
7. Place the QIAquick column in a clean 1.5ml microcentrifuge tube
8. To elute the DNA into the microcentrifuge tube, add 30 microliters of water to teh center of the QIAquick membrane, allow it to sit for 1 minute to increase the DNA concentration, then centrifuge for 1 minute.

Now the product is run thru digestion.

Digestion

1. Add 4 microliters of 10x Buffer#2 to the eluted DNA
2. Add 4 microliters of BSA to the tube
3. Add 2 microliters of the premade enyxme mix to the tube (composed of HindIII/XhoI)
4. Mix the contents ths far by setting the pipet to 25 microliters and gently pipetting up and down 2-3 times

The primers have the sites for the restriction enxyme, and will cut the sample there. Later, it will be mixed with similarly restricted bacterial plasmids. Because both the plasmid and the sample DNA has been cut in the same with, they will be able to 'stick' to one antoher, and thus the mutant DNA will recombine into the bacterial plasmid, thus priducing a recombinant, transgenic, mutant genome.