Alice:LabNotes/2008-10-3: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Zsakura2
(New page: Experiment: Elongating Cosmic Probe length template used is the probe after circleHelper insertion (see 9-15-08 labnote), which is 160bp long. The nanodrop result is 31.8 ng/ul, which ...)
 
>Zsakura2
No edit summary
Line 1: Line 1:
Experiment: Elongating Cosmic Probe length
==Experiment: Elongating Cosmic Probe length==
   
   
  template used is the probe after circleHelper insertion (see 9-15-08 labnote), which is 160bp long. The nanodrop result
  template used is the probe after circleHelper insertion (see 9-15-08 labnote), which is 160bp long. The nanodrop result
Line 9: Line 9:
  When using 10ul of template, we obtain 10ul x 0.6023uM = 6.026umol. so the amount of both spacer oligo needed is  
  When using 10ul of template, we obtain 10ul x 0.6023uM = 6.026umol. so the amount of both spacer oligo needed is  
  6.026umol/10uM= 0.6ul.
  6.026umol/10uM= 0.6ul.
Prepare the oligo: combining 0.6ul of reverse and insertion oligo, which is a total of 1.2 ul.
                    denature at 95C for 5min, and then anneal at 65C for 20min.
Prepare the probe: take out 10ul of template (product after circlehelper insertion and purification)
                    95C for 5 min, then 65C for 1 min, add 1ul of 10x ampligase buffer, 0.5ul of ampligase, 1.2 of primer
                    anneal at 65C for 30 min.

Revision as of 23:53, 5 October 2008

Experiment: Elongating Cosmic Probe length

template used is the probe after circleHelper insertion (see 9-15-08 labnote), which is 160bp long. The nanodrop result
is 31.8 ng/ul, which is 31.8 ng/(160x330) = 602.3 nM.
The amount of insertion spacer oligo ordered was 2nmol, and the reverse arm was 4.8nmol. Diluting them using 200ul and 480ul
of ddH2O respectively yields final concentration of 10uM.
When using 10ul of template, we obtain 10ul x 0.6023uM = 6.026umol. so the amount of both spacer oligo needed is 
6.026umol/10uM= 0.6ul.
Prepare the oligo: combining 0.6ul of reverse and insertion oligo, which is a total of 1.2 ul.
                   denature at 95C for 5min, and then anneal at 65C for 20min.
Prepare the probe: take out 10ul of template (product after circlehelper insertion and purification)
                   95C for 5 min, then 65C for 1 min, add 1ul of 10x ampligase buffer, 0.5ul of ampligase, 1.2 of primer
                   anneal at 65C for 30 min.