mutagenesis question

Hong Dang hdang at cns.neusc.bcm.tmc.edu
Fri May 26 12:09:28 EST 1995


In article <95144.215132MAYL at QUCDN.QueensU.CA>, <MAYL at QUCDN.QueensU.CA> writes:
|> We usually do this in the following manner:
|> 
|> Design 4 primers,
|> 1 5' to your mutation (a)
|> 1 3' to your mutation (d)
|> 2 at your mutation, going in opposite 'directions' (b and c)
|> 
|> For this to work, the primers at the 5' and 3' sites have
|> to contain unique restriction sites, and the mutation has
|> to create a new restriction site...
|> 
|> 
|>   a                b
|> ------>         ----->
|> ===================x==================
|>                 <-----       <------
|>                     c              d
|> 
|> PCR out two fragements, one using Primers a and c, the other
|> using primers b and d. Cut the products with the appropriate
|> enzymes, and ligate into a vector (3 part ligations are *fun*)
|> 
|> then sequence to check for errors. Or subclone each fragment,
|> sequence, then cut and ligate.
|> 
|> Good luck:)
|> 
|> Lorraine

We used 3 primers, a, b, and d. Do PCR with b and d, then a second PCR
with a and the product from the 1st PCR, with Pfu polymerase for the
1st reaction (works well at least under 500 bp), and 5% DMSO. Then subclone
the final product, works quite well for us.

Hong



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