AlanFung:LabNotes/Probe/2012-5-14

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Revision as of 21:29, 14 May 2012 by >Alan6017518 (Created page with "==Quantification of DNA using denaturing gel and a quantitative ladder== Combine all tubes into a 1.5mL tube 1. Add 4uL of 5ng/uL 90-mer, 4ul of H2O and 8 uL of 2x TBE-Urea b...")
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Quantification of DNA using denaturing gel and a quantitative ladder

Combine all tubes into a 1.5mL tube

1. Add 4uL of 5ng/uL 90-mer, 4ul of H2O and 8 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.