发信人: Tharu (我是恐龙我怕谁), 信区: Biology
标 题: Manual-Re: who has experience with PCR-based mutangenesis?
发信站: The unknown SPACE (Fri Sep 13 13:03:45 2002) WWW-POST

You don't need 5' phosphorylated primers if you are only going to use one set
of complementary primers. If you are using multiple primers to introduce
multiple changes simultaneously, then you need to phosphorylate the 5'.
Please refer to the details in their manuals:
http://www.stratagene.com/manuals/200518.pdf
Here are a few personal comments on the Stratagene's Quickchange system:
1. NO NEED to buy their kit, it's very over-priced. You need to get a high
fidelity DNA polymerase (for example, their pfuTurbo), and Dpn I. The rest you
probably already have in your lab.
2. NO NEED to PAGE-purify your primers. Of course it doesn't hurt, but in my
experience, desalted primers are already good enough.
3. Love it for the high efficiency. Always worked the very first time. I've
pushed it to insert 3-nt and substitute 1 in one set of primers in a 12-kb
plasmid, and its efficiency is 50%. A lot higher than what they dare to claim
in their manual and on their website. Hehe.
4. Their new Multi-change system is plainly awful. Maybe it's because of the
low transformation efficiency of ssDNA. A new grad student in the lab has
tried to get it to work for months and it still doesn't. So try to stay away
from it.
Ok, hope it helps and good luck!
【 在 royluo (roy) 的大作中提到: 】
: maybe not necessary. I never ask for extra 5' phosphorylation
: when I order primers from Invitrogen, and it works pretty well.
:
: 【 在 golem (狗愣小岩) 的大作中提到: 】
: : do I need phosphorylated 5' ended primers?
: : 【 在 royluo (roy) 的大作中提到: 】
: : : sorry. I mean the two primers are complementary to each other.
:
:


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※ 修改:·Tharu 於 Sep 13 13:03:45 修改本文·[FROM: 129.187.]
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 129.187.]