AlanFung:LabNotes/Probe/2012-5-14: Difference between revisions

From ZhangLabWiki
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.
[[Media:ZhangLab_2 2012-05-14 15hr 46min.jpg|400px]]
 
[[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