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