AlanFung:LabNotes/Probe/2012-5-14: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
(One intermediate revision by the same user not shown) | |||
Line 10: | Line 10: | ||
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. | 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. | 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. | ||
[[ | |||
[[File:ZhangLab_2 2012-05-14 15hr 46min.jpg|400px]] | |||
*Using the linear trendline we obtain the following formula, where x is ng and y is the intensity | |||
*y = 546.85x + 2758.3 | |||
*the 1ul probe's intensity is at 18876.28 which converts to 29.47ng | |||
*the 2ul probe's intensity is at 22667.29 which converts to 36.41ng |
Latest revision as of 18:54, 15 May 2012
Quantification of DNA using denaturing gel and a quantitative ladder[edit]
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 1. Add 4ul of low mass ladder, 1ul 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 (2uL sample + 8 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.
File:ZhangLab 2 2012-05-14 15hr 46min.jpg
- Using the linear trendline we obtain the following formula, where x is ng and y is the intensity
- y = 546.85x + 2758.3
- the 1ul probe's intensity is at 18876.28 which converts to 29.47ng
- the 2ul probe's intensity is at 22667.29 which converts to 36.41ng