1.0 Purpose
1.1 This protocol may be used for the linearization and purification of the shuttle plasmid.
2.0 Materials
2.1 Equipment
DNA Gel Electrophoresis Apparatus
Heating block set at 65 ºC
Centrifuge
-20 ºC Freezer
UV Spec
2.2 Supplies
Restriction Enzymes
Buffered saturated phenol solution
Chloroform
NaAc
Ethanol
DNase, RNase free water
3.0 Procedure
3.1 Linearization
3.1.1 Linearize shuttle plasmid with either PmeI, NheI, SwaI, or SfiI. Make sure the enzyme you choose does not cut in your insert.
3.1.2 Run sample on gel to confirm complete linearization.
3.1.3 Heat inactivate your sample in the heat block at 65ºC for 20minutes.
3.2 Purification of Plasmid
3.2.1 Bring your buffer saturated phenol (in fridge) to room temp and gather your necessary ingredients: chloroform w/ ampho alcohol, and 3M NaAc
3.2.2 Add an equal volume of buffer saturated phenol to your tube and shake vigorously for about 3 min.
3.2.3 Centrifuge at 14,000 rpm in the microfuge for 5 minutes.
3.2.4 Transfer the upper layer to a new tube (try not to get any of the phenol)
3.2.5 Add an equal volume of chloroform to the sample and shake vigorously.
3.2.6 Centrifuge at 14,000 rpm in the microfuge for 5 minutes.
3.2.7 Add a 1/10 volume of 3M NaAc pH 5.5 to the sample (ie. If you have 500ul, then add 50ul NaAc).
3.2.8 Mix and place at –20 C for 30minutes
3.2.9 Pellet sample in microfuge at 14,000 rpm for 10 minutes.
3.2.10 Check to see that there is a pellet.
3.2.11 Remove the supernatant after you see a pellet. Rinse with 1ml of 70%EtOH and quick spin for 3 min at 14,000. Remove the sup after you see a pellet.
3.2.12 Resuspend the pellet in DNAse RNAse free H2O..
3.2.13 Spec for concentration
