Editing Taq-Citraconylation-2

Procedure

9.1.&nbsp; Polymerase Modification<br>Note: This procedure describes the quantities of reagents and materials used to modify 100 mL of Taq polymerase. If larger or smaller volumes are needed, the quantities of each reagent can be increased or decreased accordingly.<br>9.1.1&nbsp; Test Modification â A test modification can be done using 10 mL of the diluted Taq polymerase (Step 9.1.3). This is optional and may need to be done by inexperience operator. Ask your supervisor to determine if it should be performed. For the test modification, complete Step 9.1.2 through 9.2.2.4 and compare the results to Appendix 10.3. If the modification reaction did not appear to work, report it to your supervisor. If you are not going to perform this test modification, skip to Step 9.1.2 and modify the desired volume.<br>9.1.2&nbsp; Measure the OD280 of the purified Taq polymerase that is to be modified and record it. Save 1 mL of polymerase and label it âunmod Taqâ along with date.<br>9.1.3&nbsp; Dilute and aliquot of Taq to and OD280 of 1.8 ot 2.1 with 1x Buffer G (if required) such that the final volume is 100 mL. Record the volumes. Measure the final OD280 and record it. <br>9.1.4&nbsp;&nbsp; Place the 100 mL of diluted Taq polymerase in a 200-500 mL bottle and place a magnetic bar in it.<br>9.1.5&nbsp;&nbsp; Place the Taq on a magnetic stir plate at room temperature and begin mixing the solution.<br>9.1.6&nbsp;&nbsp; Add 10 mL of 1M Na2HPO4 all at once and record the volume.<br>9.1.7&nbsp;&nbsp; Add 30 uL of citraconic anhydride and record the volume.<br>9.1.8&nbsp;&nbsp; During this 15 min incubation, measure the pH of the solution using pH paper and record it. If the pH is not 8-9 use 0.5M NaOH to adjust the pH to within this range and record it.<br>9.1.9&nbsp;&nbsp; Repeat Steps 9.1.7 and 9.1.8 three additional times. (Four 15 min incubations for a total of 1 hr).<br>9.1.10&nbsp; Contunue the stirring and incubating at room temperature for another 30 min.<br>9.1.11&nbsp; During this incubation, prepare 4 L of 20mM Tris pH 8.3 by diluting 80 mL of 1M Tris pH 8.3 in 3920 of ddH2O. Place this solution at 4oC.<br>9.1.12&nbsp; After the 30 min incubation is complete, transfer the contents of the bottle to 12-30 mL capacity dialysis cassettes and place them in the 4 L of 20 mM Tris pH 8.3 prepared in Step 9.1.11. Add a stir bar to the dalysis buffer and place the dialysis beaker on a stir plate at 4 oC. Dialyze the sample for 12-20 hr.<br>9.1.13&nbsp; Transfer the contents of the dialysis cassttes to labeled tubes and store them at 4 oC<br>

[[Category:Protocols]]