Alice:LabNotes/2008-4-14
Jump to navigation
Jump to search
padlock probe library preparation[edit]
Step 9: Precipitation
1. Add 0.1x of 3M sodium acetate and 2.5x 100% ethanol. 2. Add 1uL Glycoblue so that one can precipitate better and see the DNA. 3. Freeze in -70C for 20 min, spin at 14,000 rpm for 15 min at 4C, discard supernatant with pipette. 4. Add 500 uL of 75% ethanol to wash DNA, spin at 8000 rpm for 5 min at 4C, discard supernatant with
first large tip and then small tip to remove as much liquid as possible. 5. Let dry in hood ~ 10 min. 6. Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.
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.
File:ZhangLab 2 2008-04-14 18hr 01min--1.JPEG
The concentration is 14.98 ng/ul. Yield is 14.98 ng/ul x 20ul= 299.6 ng