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Richard Lab:Amplified insert assembly: Difference between revisions
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Richard Lab:Amplified insert assembly (view source)
Revision as of 15:09, 25 January 2011
, 25 January 2011→Procedure
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#[[Richard Lab:Restriction Digest|Digest]] the "vector" for 2 hours. | #[[Richard Lab:Restriction Digest|Digest]] the "vector" for 2 hours. | ||
#Purify the PCR product using a kit or [[Ethanol precipitation of nucleic acids|this protocol]]. | #Purify the PCR product using a kit or [[Ethanol precipitation of nucleic acids|this protocol]]. | ||
#[[Richard Lab:Restriction Digest|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases). | #[[Richard Lab:Restriction Digest|Digest]] insert for 1 hour (adding [http://www.neb.com/nebecomm/products/productR0176.asp DpnI] along with the other restriction endonucleases). | ||
#Add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done. | #Add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done. | ||
#Kill all reactions by incubating for 20 mins at 80°C. | #Kill all reactions by incubating for 20 mins at 80°C. | ||
