how to amplify fragment from the plasmid - (Sep/21/2005 )
i will amplify a 9.4kb fragment from the plasmid, but i still don't know which aspects i should pay attention to during the pcr . which polymerase do you usually use ? is there any kit for long pcr?
i have amplified several times , but no band was seen on the gel.
hi phusion or pfu polymerases are more precise as the famous taq, and also more processive.
I've successfully used 4 or 5 years ago a kit to amplify a 13kb fragment. It was the 20kbplus PCR system from roche and process was not that long...
There's plenty of enzymes for long PCR, every supplier has at least one. Pay attention to their protocols and go ahead.
You have to worry about introducing errors in your sequence by using PCR. For such a long sequence you probably will have one or two changes. I recommend requencing this in the coding regions you are using to prevent trouble down the road. It can cost you a year of your life figuring out why your protein is not expressed!
BTW, The Phusion enzyme is great and has a very low error rate. Just need to play around with the PCR conditions because it behaves differently than standard taq