e***o
发帖数: 344 | 1 各位做酵母转化时一般一个细胞里能转进多少个不同的plasmid，等酵母涨到比较大的
克隆时估计还有多少种plasmid
不知有人是否注意到这个没有，谢谢 |
w**t
发帖数: 52 | 2 All depend on your transformation efficiency. If you are talking about co-
transforming different plasmids, I will say it is not easy to co-transform 4
plasmids at the same time. And if you are doing library transformation,
there will be many different plasmids in one cell. The traditional way to purify
your transformants is streaking the single colony for 2-3 times. |
e***o
发帖数: 344 | 3 谢谢回复！我准备做library transformation，看来会有很多不同的质粒了。你的意思
是划几次线酵母增值就会把质粒稀释掉？如果晒库的话，划线的工作量还是有点大，不
知道直接洗下来，重新铺板是不是一样有效果？ |
w**t
发帖数: 52 | 4 Are you going to do Y2H screening? If so, only those interact could grow on
selective plates (SCM-L-T-H). You could further purify your candidates by streaking before you recover the
plasmids. |
e***o
发帖数: 344 | 5 谢谢。 我准备做超大量的screening, 如果一个一个划线的话，可能工作量太大。不知
道直接把colony洗下来，重新铺板或是用copy plate的办法，也能dilute copy number
到一个或2个copy？
btw, 你知道酵母里面那个inducible promoter比较好用？比如leaking expression比
较少？ |
l**********1
发帖数: 5204 | 6 让你老板买个半自动或全自动工作站做Y-2-H 不就得拉
或借用同校里的其它单元的那设备
没听说过欧美高通量Y-2-H 筛选用一双手做的
半自动工作站 96 well
please below link:
//www.tecan.com/platform/content/element/5135/Tecan_Journal_09_01.pdf
for one
France developed a semi- automated yeast two-hybrid system with the Te-
MOTM 96 Multichannel Pipetting Option from Tecan, halving the time taken to
obtain reproducible results from six to three days.
if try Y-1-H
全自动工作站 384 well
please refer last Oct. Nature Methods two papers:
//www.ncbi.nlm.nih.gov/pubmed/22037705
//www.ncbi.nlm.nih.gov/pubmed/22037703
number
【在 e***o 的大作中提到】
: 谢谢。 我准备做超大量的screening, 如果一个一个划线的话，可能工作量太大。不知
: 道直接把colony洗下来，重新铺板或是用copy plate的办法，也能dilute copy number
: 到一个或2个copy？
: btw, 你知道酵母里面那个inducible promoter比较好用？比如leaking expression比
: 较少？
|
w**t
发帖数: 52 | 7 Are you going to do a genetic screening?
For the inducible promoter, I recommand MET3 and GALS, both are very tight. |
e***o
发帖数: 344 | 8 thanks a lot for you guys' inspiring reply. I am indeed going to do
screening. But I am developing a new method which requires 1-2 copies per
cell and can not use the automated station. Hopefully washing and replating
the colonies will dilute the copy number just like streaking.
BTW, do you guys know why the copy number decreased after streaking? |
w**t
发帖数: 52 | 9 The streaking do not 'dilute' the plasmid, but simply purify the colonies.
The copy number of the plasmid mostly depend on your replication origin (
never use 2u origin if you want a low copy), and whether the plasmid contain
centromere (like CEN6 on pRS416). Usually the centromere sequence can limit
the copy number of your plasmid.
I don't think you need to wash or replica your plates. If you don't want to
have too many plasmids in one cell, just simply reduce the transformation
efficiency and do more transformation. Or you can culture your transformants
for 1~2 generations before you plate them.
BTW, you should have some ways to select your candidates. Then culture the
cells under selective pressure should allow you to enrich the plasmid you
want. |
l**********1
发帖数: 5204 | 10 En i see.
if likes ASIMO robot can do that task almost Lab technician plus some share
PD should go to taxi driver from then.. joke just.
replating
【在 e***o 的大作中提到】
: thanks a lot for you guys' inspiring reply. I am indeed going to do
: screening. But I am developing a new method which requires 1-2 copies per
: cell and can not use the automated station. Hopefully washing and replating
: the colonies will dilute the copy number just like streaking.
: BTW, do you guys know why the copy number decreased after streaking?
|
c*******s
发帖数: 737 | |
