Alice:LabNotes/2008-2-11
Experiment 1: Padlock library preparation
Step 11: Quantification of DNA using denaturing gel and a quantitative ladder
1. Add 8uL of 2ng/uL 90-mer and 8 uL of 2x TBE-Urea buffer
2. Dilute sample 10x (2uL sample + 8 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE-Urea buffer.
3. 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.
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, 1.25 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.
The image below is the gel:
File:ZhangLab 2 2008-02-12 12hr 58min.jpeg
There was a small blue pallette after I finished ethanol precipitation, so the gel should show stronger bands. However, the bands of the sample are very weak and almost hardly can see.