GP43 DNA Polymerase

The T4 DNA Polymerase is responsible for catalyzing the incorporation of deoxynucleotides in the 5’ 3’ direction. The wild type T4 polymerase has a strong 3’ to 5’ exonuclease activity, however the polymerase produced in the lab is an exonuclease deficient mutant.

Contents

1. Notes
2. Transform
3. Express
4. Purify
5. Analysis
6. Notes on Activity

1. Notes

• GP43 is 112KDa
• Plasmid given by collaborator Scott Nelson

2. Transform

1. Transform SNgp43 (Scott Nelson’s gp43 plasmid) into BL21(DE3) cells.
   1. Use 50 uL of Bl21(DE3) cells and add 1 uL of 20ng/uL of SNgp43.
   2. Flick 5 times to mix and place on ice for 5 minutes.
   3. Place the cells in a 42C water for 42 seconds.
   4. Remove and immediately place on ice for 2 minutes.
   5. Add the cells to 500 uL of SOC.
   6. Allow to grow in a shaker at 37C, 250rpm for 1 hour.
   7. Remove and plate 250 uL of culture onto an LB-Kan plate.

3. Express

1. Take a single colony from the plate and inoculate an overnight of 100 mL with a 50 ug/mL concentration of Kanamycin.
2. Use 10 mL of overnight to inoculate 3L of LB and culture at 37C, 250RPM.
3. Grow until the O.D. 600 is equal to 0.6-1.0.
4. Cool cultures to 16C then inoculate the cultures with a concentration of 0.4 mM IPTG.
5. Allow cultures to grow overnight at 12C in shaking incubator for 18-24 hours.
6. Harvest by centrifuging for 10 minutes at 5000 rpm.
7. Decant the supernatant and keep the pellet Do this on ice in the cold room.
8. Resuspend pellets in 70 mL with resuspension buffer:
   • 50 mM Tris pH 7.5
   • 500 mM NaCl
   • 1 mM EDTA
   • 10% Sucrose
   • 1 mM PMSF (protease inhibitor)
9. Separate the cell suspension into (2)-45ml aliquots.
10. Flash freeze cell suspensions in liquid nitrogen and store in -80C until ready to use.

4. Purify

Do as much of this procedure on ice in the cold room as possible

1. Defrost the cells on ice.
2. Sonicate at 75% amplitude for bursts of 15 seconds with rests of 90 seconds for a total sonication time of 90 seconds.
   • The lysate should flow easily if cells are broken
3. Centrifuge the cells in the ultracentrifuge at 35K for 35 minutes at 4C.
4. Collect the supernatant and discard the pellet.
   • Clean the centrifuge tubes
5. Set aside a 1 mL sample of clarified lysate for analysis on an SDS page gel.
6. Load the rest of the clarified lysate onto a 5 mL chitin column equilibrated with 50 mM Tris pH 7.5, 100 mM NaCl and 1 mM EDTA (Buffer A).
7. Once loaded, wash the column with 1L of 50 mM Tris pH 7.5, 1M NaCl and 1 mM EDTA (Buffer B).
   • Set aside 20 mL of Buffer B for cleavage of protein from the column
8. Add DTT to a concentration of 50 mM to the 20 mL of Buffer B to make it cleavage buffer.
9. Add the 20 mL of cleavage buffer to the column and allow 10 mL of it to drip through.
10. Close the stopcock and let the other 10 mL of cleavage buffer incubate with the beads overnight.
11. In the morning, collect 1 mL fractions.
12. Analyze all collected fractions on a 10-12% SDS-PAGE.
13. Pool the purified fractions together and dialyze overnight into:
    • 20 mM Tris pH 7.5
    • 150 mM NaCl
    • 10% glycerol
    • Check pH!
14. Aliquot protein into 10 uL samples into 0.5 uL Eppendorf tubes.
15. Flash freeze protein aliquots using liquid nitrogen.
16. Store at -80C.

5. Analysis

1. Check the DNA to protein ratio (260/280) using the nanodrop.
2. Check the quantity of protein using a Bradford Assay.

6. Notes on Activity

• (from Scott Nelson) gp43 is fairly stable and can be re-frozen upon use; however, make sure that it is always on ice if thawed!