Problem of Plasmid Preparation - (Aug/27/2008 )
A gene has been cloned into pIRES2-EGFP. This plasmid can be prepared from DH5a.
However, when a mutation is introduced into the gene, the mutant can not be prepared from the same competent cells. The transformed cells can not be lysised by Buffer II.
(I am sure that the buffer is no problem)
Thank you!
I don't see any reason why this would be the case 
Did you try to do a crude miniprep first on thoses clones to check ?
However, when a mutation is introduced into the gene, the mutant can not be prepared from the same competent cells. The transformed cells can not be lysised by Buffer II.
(I am sure that the buffer is no problem)
Thank you!
Want to understand more before figuring out the problem
what do you mean by "can not be lysed by Buffer II" ?
Normally after buffer I, you will get a turbid/cloudy suspension
Then adding buffer II the suspension will clear up
What did you see after adding buffer II that makes you think the cells cannot be lysed??
Did you try to do a crude miniprep first on thoses clones to check ?Yes, I have tried crude miniprep.
However, when a mutation is introduced into the gene, the mutant can not be prepared from the same competent cells. The transformed cells can not be lysised by Buffer II.
(I am sure that the buffer is no problem)
Thank you!
Want to understand more before figuring out the problem
what do you mean by "can not be lysed by Buffer II" ?
Normally after buffer I, you will get a turbid/cloudy suspension
Then adding buffer II the suspension will clear up
What did you see after adding buffer II that makes you think the cells cannot be lysed??
The suspension just can not be clear after adding Buffer II.
I have sent it to the Company for sequencing, and they also can`t prepare the DNA.
Might the buffer II be old? Atmospheric carbon dioxide might have reacted with it.
Anyhow how about trying the following; First freezing your cells, then thaw them in a lysozyme solution (at 37 C, 15-20 minutes), followed by lysis with freshly made NaOH/SDS (buffer II solution). This protocol is usually used when handling large Ecoli paste, but it should be enough to break your harden e coli walls
Anyhow how about trying the following; First freezing your cells, then thaw them in a lysozyme solution (at 37 C, 15-20 minutes), followed by lysis with freshly made NaOH/SDS (buffer II solution). This protocol is usually used when handling large Ecoli paste, but it should be enough to break your harden e coli walls
Thank you for your reply and I`ll try your method.
(I think the BufferII is ok because other plasmids can be prepared by this buffer)
Sounds like contamination of a different bacterium.