RNA Reaction - Cycle Runoffs

Jeff Lawrence

1. Kinase an oligonucleotide which extends toward the promoter of interest.

2. Add 100 µL of a fresh overnight culture to 10 mL of the appropriate medium in a 125 mL Klett flask. Grow with shaking until mid-log phase, about a net Klett of 75.

3. Preheat plastic capillary tube sealer.

4. Transfer 1.5 mL cells to a chilled microcentrifuge tube. Spin for 2 min at 4°. Remove supernatant by aspiration.

5. Resuspend pellet in 1 mL ice-cold ddH2O. Spin for 2 min at 4°. Remove supernatant by aspiration.

6. Resuspend pellet in 1 mL ice-cold ddH2O. Place on ice.

7. Prepare runoff reaction mix. Use the full 20 µ:L as some evaporation occurs during extension
	2.0 µ:L	Rinsed cells
	2.0 µ:L	1 µ:M kinased oligonucleotide, for a total of 2 pmol
	2.0 µ:L	10X RT buffer (Promega)
	2.0 µ:L	10X MnCl2 (Promega)
	2.0 µ:L 	8 mM dNTPs (2 mM each)
	0.5 µ:L	Tth DNA polymerase (Promega)
	9.5 µ:L	ddH2O
	20 µ:L	Total Volume
8. Load reaction into a plastic capillary tube and seal one end. Remove the tube from the syringe and seal the other end. Seal all tubes, storing them on ice.

9. Place tubes in an Idaho Technologies Thermal Cycler. Program the following profile:
	Cell lysis	94°	45 sec
	Denaturation	94°	2 sec
	Annealing	60°	2 sec
	Extension	72°	2 min
	Slope  		S=6
	Cycles 		C=96
10. Cycle 4 replicate of each reaction. Remove one replicate after 12 cycles, 24 cycles, 48 cycles, and 96 cycles.

10. Decant and dry to 5 µ:L final volume. Add 5 µ:L Stop Dye. Heat to 70° for 2 min and keep on ice.

11. Electrophorese 1 µ:L on a polyacrylamide gel next to a DNA sequence generated with the same primer.

12. Verify that the band of interest is amplified arithmetically, halving in intensity from 96>48>24>12 cycles. PCR artifact bands will increase geometrically and will appear suddenly.

Last Update: Thursday, 19-Jun-2014 11:50:18 PDT
This page has been viewed 66 times.
Eric Kofoid eckofoid at ucdavis.edu