DNA ligation: Difference between revisions

Revision as of 11:07, 28 April 2005

Added By

Ligation Mix

6:1 Molar ratio of insert to vector ([math]\displaystyle{ \sim }[/math]10ng vector)
X[math]\displaystyle{ \mu }[/math]L dd[math]\displaystyle{ \rm{H_2O} }[/math] to bring the total to 10[math]\displaystyle{ \mu }[/math]L

Calculating Insert and Vector Amounts

Insert Mass =[math]\displaystyle{ 6\times\left[\frac{\rm{Insert\ Length}}{\rm{Vector\ Length}}\right]\times \rm{Vector\ Mass} }[/math]

Reaction Conditions

Let the 10 [math]\displaystyle{ \mu }[/math]L solution sit at [math]\displaystyle{ 22.5^o }[/math]C for 30min
Denature the ligase at [math]\displaystyle{ 65^o }[/math]C for 10min
Dialyze for 20 minutes if electroporating
Use disks shiny side up
Store at -20C

@@ Line 6: / Line 6: @@
 *Example - 10ul mix
-*1.0 $\mu$L 10X T4 ligase buffer
+*1.0 <math>\mu</math>L 10X T4 ligase buffer
-*0.5 $\mu$L T4 Ligase
+*0.5 <math>\mu</math>L T4 Ligase
-*6:1 Molar ratio of insert to vector ($\sim$10ng vector)
+*6:1 Molar ratio of insert to vector (<math>\sim</math>10ng vector)
-*X$\mu$l ddH$_2$O to bring the total to 10$\mu$l
+*X<math>\mu</math>L dd<math>\rm{H_2O}</math> to bring the total to 10<math>\mu</math>L
 '''Calculating Insert and Vector Amounts'''
-<math>
+Insert Mass =<math> 6\times\left[\frac{\rm{Insert\ Length}}{\rm{Vector\ Length}}\right]\times \rm{Vector\ Mass} </math>
-Insert Mass = 6\times\left[\frac{Insert Length}{Vector Length}\right]\times Vector Mass
-</math>
 '''Reaction Conditions'''
-*Let the 10 $\mu$l solution sit at 22.5$^o$C for 30min
+*Let the 10 <math>\mu</math>L solution sit at <math>22.5^o</math>C for 30min
-*Denature the ligase at 65$^o$C for 10min
+*Denature the ligase at <math>65^o</math>C for 10min
 *Dialyze for 20 minutes if electroporating
 *Use disks shiny side up
 *Store at -20C