p*******n
发帖数: 6 | 1 我刚开始做金葡菌的致病因子，要用phage从一种strain上载入一个mutant DNA片段导
入另一个strain。用的phage是transducing phage，据提供者说high frequency of
lysogenization。我用plaques提取lytic phage，然后和recipient混合，incubate 20
分钟以后立即稀释、涂板，结果没有一个带resistant marker的colony长出来。
我的实验方法可能会出什么问题？
这种phage是不是主要进行specialized transduction？收集lysogen然后transduce是
不是可行的方法？
谢谢各位。 |
A********2
发帖数: 107 | 2 If I understood your question correctly, you have inserted a Ab resistance
gene (linked to the mutation) into the mutant chr and were trying to move it
into wild type strain. Why not just make a phage lysate of your mutant
strain and then transduce the wild type strain. |
p*******n
发帖数: 6 | 3 yes, you got my question.
The mutant comes with an Ab-resistant marker.
I am culture phage with mutant strain, and then collect the phage from
plaques by homogenizing the agar chunks. However, these phage do not produce
Ab resistant colonies on the Ab-selective plates.
I am wondering if only the lysogens can carry the mutant gene with Ab
resistance marker.
Thank you for replying me.
it
【在 A********2 的大作中提到】
: If I understood your question correctly, you have inserted a Ab resistance
: gene (linked to the mutation) into the mutant chr and were trying to move it
: into wild type strain. Why not just make a phage lysate of your mutant
: strain and then transduce the wild type strain.
|
A********2
发帖数: 107 | 4 My suggestion is:
1. Test if your recipient strain is phage sensitive
2. Determine how many PFU per ml in your lysate by phage titration assay and
use the appropriate amount of lysate in the transduction. |
