AlanFung:LabNotes/Probe/2012-5-14: Difference between revisions
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==Quantification of DNA using denaturing gel and a quantitative ladder== | ==Quantification of DNA using denaturing gel and a quantitative ladder== | ||
Combine all tubes into a 1.5mL tube | Combine all tubes into a 1.5mL tube | ||
1. Add 1ul of low mass ladder, 4ul of H2O and | 1. Add 1ul of low mass ladder, 4ul of H2O and 5 uL of 2x TBE-Urea buffer | ||
1. Add 2ul of low mass ladder, 3ul of H2O and | 1. Add 2ul of low mass ladder, 3ul of H2O and 5 uL of 2x TBE-Urea buffer | ||
2. Dilute sample 10x (1uL sample + 9 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE buffer | 2. Dilute sample 10x (1uL sample + 9 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE buffer | ||
3. Denature the DNA with loading dye on thermalcycler 75C for 7 min, quickly transfer to cooling rack, wait for 1 min. | 3. Denature the DNA with loading dye on thermalcycler 75C for 7 min, quickly transfer to cooling rack, wait for 1 min. |
Revision as of 21:33, 14 May 2012
Quantification of DNA using denaturing gel and a quantitative ladder
Combine all tubes into a 1.5mL tube
1. Add 1ul of low mass ladder, 4ul of H2O and 5 uL of 2x TBE-Urea buffer 1. Add 2ul of low mass ladder, 3ul of H2O and 5 uL of 2x TBE-Urea buffer 2. Dilute sample 10x (1uL sample + 9 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE buffer 3. Denature the DNA with loading dye on thermalcycler 75C for 7 min, quickly transfer to cooling rack, wait for 1 min. 4. Use 6% 10 well TBU gel, add 0.5x TBE buffer, flush the wells very well to remove all urea. 5. Pre-run the gel at 200V for 10 min, then load the samples into the wells, run at 200V for 30 min. Load the ladder as 8, 4, 2, 1 uL, and load the samples as 10, 5, 2.5 uL. 6. Stain the gel with 5 uL SYBR gold in ~100 mL of TBE in clean tray. Place the tray on an orbital shaker for 5 min. Then quantify the gel to calculate the % yield.