SOS!--PCR restriction cut

Dr. Duncan Clark duncan at genesys.demon.co.uk
Wed Mar 20 04:37:49 EST 1996


In article <Pine.SOL.3.90.960318103519.20921A-100000 at uxmal>, Edgar
Valencia <edgar at cifn.unam.mx> 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
>orientation.

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.

Duncan
DNAmp Ltd.
Licensed for PCR enzyme Production
--------------------------------------------------------------------------------
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sliced from time to time....
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