Alice:LabNotes/2008-11-4: Difference between revisions

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>Zsakura2
>Zsakura2
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==diagnosing spacer annealing and insertion efficiency==
==diagnosing spacer annealing and insertion efficiency==
  <font color=green>For the product and procedure from previous steps, please follow this link (10/14/08 LabNote):  [[http://genome-tech.ucsd.edu/LabNotes/index.php/Alice:LabNotes/2008-10-14]]</font>
  <font color=green>For the product and procedure from previous steps, please follow this link (10/14/08 LabNote):  [[http://genome-tech.ucsd.edu/LabNotes/index.php/Alice:LabNotes/2008-10-14]]</font>
  Add 1ul of spacer to 99ul of ddH2O to dilute them to 100nM
  Add 1ul of spacer to 99ul of ddH2O to dilute them to 100nM
  the final concentration of spacer is 20nM, so 20nM x 50ul =  100nM x amount needed(ul)           
  the final concentration of spacer is 20nM, so 20nM x 50ul =  100nM x amount needed(ul)           

Revision as of 22:32, 4 November 2008

diagnosing spacer annealing and insertion efficiency

For the product and procedure from previous steps, please follow this link (10/14/08 LabNote):  [[1]]
Add 1ul of spacer to 99ul of ddH2O to dilute them to 100nM
the final concentration of spacer is 20nM, so 20nM x 50ul =  100nM x amount needed(ul)           
Add 10ul of each primer, denature at 95C for 5min, and then anneal at 65C for 20min.

spacer insertion:
Repeat-reaction system 2:
template (1) 680nM                  1.5 ul 
spacer mix (100nM each)              20 ul
10x Ampligase Buffer                  5 ul
Ampligase(5U/ul)                      1 ul
ddH2O                              15.6 ul
------------------------------------------
total                                50 ul