BIOL 1027 - PRINCIPLES OF BIOLOGY HONORS
SPECIAL PROJECT ON GENE CLONING

Copyright, 2001 by R.D. Locy and J.Barbosa

        Upon completion of transformation of E.coli, if all has proceeded according to our plan we should now have cells transformed with pGEM-T Easy with our PCR product inserted into the vector. In short we have now "cloned" the gene. All that remains is to accomplish 2 things. First, we need to grow up some of our transformed cells, and stabilize them so that they can be stored indefinitely for future use. This is done by adding DMSO to an overnight culture of our cells and freezing the culture to be thawed and used in the future. The second thing we need to accomplish is to prepare plasmid DNA from our transformed culture, restrict the plasmid using specific enzymes that recognize sequences found in DNA, to liberate any insert (hopefully the same size as the original PCR product) in the plasmid. The restricted plasmid preparation is then separated by agarose gel electrophoresis as we have done before to determine the size of any inserts present in the cultures selected.

Materials:
      Transformed colonies growing Petri plates with selective medium
        Sterile tooth picks
        Test tubes containing 5 mL sterile LB liquid medium containing ampicillin
        Incubator shaker at 37 ^oC
        Sterile microfuge tubes
        Dimethyl sulfoxide (DMSO)
        Microcentrifuge
        STE Buffer - 0.1 M NaCl, 10 mM Tris pH 8.0, and 1 mM EDTA pH 8.0
        Vortex mixer

        SOLUTION I - 50 mM glucose, 25 mM Tris pH 8.0, and 10 mM EDTA pH 8.0

        SOLUTION II - 0.2 M NaOH (freshfrom 10 N NaOH stock) and 1% SDS (note add NaOH first)

        SOLUTION III - 60 mL 5 M potassium acetate, 11.5 mL glacial acetic acid, and 28.5 mL H₂O

        Phenol-Chloroform-Isoamyl-alcohol (25.24.1)
        100% Ethanol

Procedure:

1. The next day transfer 1 mL of each culture innoculated in STEP 6 with a white colony to a microfuge tube along with 70 uL DMSO. Place this mixture in the freezer –80 ºC to store for future use. Pour 1.5 mL of the remaining of the culture into another microfuge tube. Centrifuge at 12,000 x g for 30 seconds at 4 ºC. Remove the medium (supernatant) by aspiration, leaving the bacterial pellet as dry as possible. Repeat this centrifugation procedure with another 1.5 mL of the same culture.
   
   
2. Resuspend the pellet in 0.5 mL STE buffer with vigorous vortexing. Centrifuge as above and remove the supernatant. Resuspend the pellet in 100 uL of ice-cold SOLUTION I by vigorous vortexing. Add 200 uL of freshly prepared SOLUTION II. Mix the content of the tubes by inverting the tubes five times (do not vortex). Store the tubes on ice, and Add 150 uL of SOLUTION III. Close the tubes and gently vortex in an inverted position for 10 seconds to disperse solution III. Store the tubes on ice for 3-5 seconds. Centrifuge at 12,000 x g for 5 minutes at 4ºC and transfer the supernatant to a new tube.
   
   
3. Add equal volume of phenol-chloroform-isoamyl-alcohol (25.24.1) to the supernatat. Mix by vortexing, centrifuge at 12,000 x g for 4 minutes. Transfer the supernatant to a fresh tube.
   
   
4. Precipitate the double-stranded DNA with 2 volumes of 100% ethanol. Mix by vortexing. Allow the mixture to stand for 2 minutes on ice. Centrifuge at 12,000 x g for 5 minutes, and remove the supernatant by gentle aspiration. Stand the tube in a inverted position on a paper towel to allow all the fluid to drain away.
   
   
5. Rinse the DNA pellet by suspending it in 1 mL of 70% ethanol, and centrifuge as in procedure 4. Remove the supernatant as described in procedure 4, and allow the pellet to dry in the air in the tube for 10 minutes.
   
   
6. Dissolve the DNA in 40 uL of TE buffer (pH 8.0) containing DNAase free pancreatic RNAase (20ug/mL). Vortex and store at 4 or -20 ºC.
   
   
7. To analyze the DNA remove 5 uL of the DNA solution from the tube in procedure 6, and place it in a new microfuge tube.
   
   
8. Restrict the DNA using selected restriction endonucleases under appropriate restriction conditions.  Note that this procedure will differ for each of the sequences cloned.  The instructor will provide you with the specific instructions for restriction of your sample.
   
   
9. Following restriction, run a sample of the restricted DNA in agarose electrophoresis as described in STEP 4 of these procedures to analyze the size of the fragment obtained by restriction of the plasmid cloning vector..