@@ Line 64: / Line 64: @@
-'''restriction digest'''
-uL DNA
-uL buffer ( 2 for most, check  http://www.neb.com/nebecomm/DoubleDigestCalculator.asp)
-.5 uL BSA
-uL enzyme 1
-uL enzyme 2
-water to 50 uL(32.5 uL, add first)
-'''oligo assembly by PCR'''
-resuspend oligos with water, amount of water= concentration(in nm)*10 in uL
-make oligo mix with 5uL of each primer
-PCR reaction:
-uL phusion
-.5uL oligo mix
-uL first oligo
-uL last oligo
-uL buffer
-uL dnTP
-dH20 to 50uL
-'''Ligation'''
-uL insert
-uL vector
-uL T4 ligase buffer
-uL T4 ligase
-incubate at <s>37C</s> no.  **Note that NEB website recommends 16C - room temp for ligations, we do ours at RT and it works well.  1 hour.
 '''Colony PCR with Green tag'''

Team:Washington/Protocols

Revision as of 22:13, 13 September 2011