Alice:LabNotes/2008-4-23: Difference between revisions
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==Experiment 1: Padlock Library Preparation== | |||
Step 10: Quantification of DNA using denaturing gel and a quantitative ladder | |||
1. Dilute sample and 90-mer 10x (1uL sample + 9 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE-Urea buffer. | |||
2. Denature the DNA with loading dye on thermalcycler 75C for 15 min, then quickly transfer to ice, wait for 1 min. | |||
Note: Transfer to ice to keep the samples from further denaturation. | |||
3. Use 6% 10 well TBU gel, add 0.5x TBE buffer, flush the wells very well to remove all urea. | |||
4. 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 4 uL, and load the samples as 10, 5, uL. | |||
5. 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. | |||
The concentration is 9 ng/ul. Yield is 9 ng/ul x 30ul= 270 ng <br> | |||
[[Image:ZhangLab_2 2008-04-23 17hr 44min---1.jpeg]] | [[Image:ZhangLab_2 2008-04-23 17hr 44min---1.jpeg]] |
Latest revision as of 23:39, 25 April 2008
Experiment 1: Padlock Library Preparation[edit]
Step 10: Quantification of DNA using denaturing gel and a quantitative ladder
1. Dilute sample and 90-mer 10x (1uL sample + 9 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE-Urea buffer. 2. Denature the DNA with loading dye on thermalcycler 75C for 15 min, then quickly transfer to ice, wait for 1 min.
Note: Transfer to ice to keep the samples from further denaturation.
3. Use 6% 10 well TBU gel, add 0.5x TBE buffer, flush the wells very well to remove all urea. 4. 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 4 uL, and load the samples as 10, 5, uL. 5. 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.
The concentration is 9 ng/ul. Yield is 9 ng/ul x 30ul= 270 ng