SOS!--PCR restriction cut

Dr. Duncan Clark duncan at
Wed Mar 20 04:37:49 EST 1996

In article <Pine.SOL.3.90.960318103519.20921A-100000 at uxmal>, Edgar
Valencia <edgar at> writes
>Hi Wenjin Yu:
>Just a stupid question, but it happened to me. Are your restriction sites
>correctly oriented? I mean In your oligos EcoRI is 5' XXXXXXGAATTCxxxxxx 3'?
>I constructed some oligos with a "EcoRI site", added the bases at the 5'
>end to avoid cutting problems. The PCR was nice, the product has the 
>internal restriction sites it should had, but when i tried to cut with 
>EcoRI an ligate it it was impossible!. And then i saterted to modify all 
>my conditions, from vector:band rates till cleaning the PCR product and 
>vector and nothing! and then, just for curiosity I saw the restriction 
>table in a catalog and theres a enzyme, AfllI which has the recognition 
>sequence 5' CTTAAG 3', the opposite to EcoRI, so I can figure out now 
>what happened and ill try with new oligos with the EcoRI site in the rigth

Your primres have the EcoR I site correct. Do not change to CTTAAG 'cos
then it will only cut with Afl III. Your problem lies elsewhere.

DNAmp Ltd.
Licensed for PCR enzyme Production
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

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