X***n
发帖数: 366 | 1 Anyone can help?
I need pKD46 to retrieve a genomic sequence from BAC.
Do you know which company sell pKD46? |
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发帖数: 1 | 2 想用lamda red在BL21中敲一个基因，按照protocol，把pKD46转入BL21，很奇怪的是不
管用化转还是电转，涂板后都没有长出来，但是转其他的质粒都没有问题。对pKD46跑
胶和单酶切都没有问题，按照protocol所有incubation的温度都是30度。同时还试过E.
coli 另外的菌株BW25113也是出现同样问题。有人遇到过质粒转成这样的情况吗？多谢
！ |
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X***n
发帖数: 366 | 3 Thank you very much!
I really appreciate your help! |
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X***n
发帖数: 366 | 4 how about using pKD46 to flank the gene you want to knock out with FRT,then
insert FLP gene downstream the promoter that activate at the specific time
point?
I don't know details :) |
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X***n
发帖数: 366 | 5 I cloned 400-500 bp miniarms cloned into ptdTomato-C1, and then linearized
it with EcoRI, so that the backbone was flanked by miniarms on both ends. I
electroporated the linearized plasmid into the induced Red recombinases in
SW102 carrying my BAC. All the clones turns to be empty. I succeed in the
other two using pKD46 system but failed in this one. And now I also fail
using SW102 system. What's the most possible reason? Thanks in advance. |
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X***n
发帖数: 366 | 6 如果这12基因在细菌基因组中是连在一起的，直接用recombineering拉下来就行，搜一
下pKD46就行。 |
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X***n
发帖数: 366 | 7 google recombineering, pkd46
Recombineering‐Based Procedure for Creating BAC Transgene Constructs for
Animals and Cell Lines
Steven M. Hollenback1, Suzanne Lyman1, JrGang Cheng1
1 Neuroscience Center, UNC‐Chapel Hill, Chapel Hill, North Carolina
Publication Name: Current Protocols in Molecular Biology
Unit Number: Unit 23.14
DOI: 10.1002/0471142727.mb2314s95
Online Posting Date: July, 2011 |
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j****n
发帖数: 3370 | 8 怀疑plasmid有问题
好奇为啥验证用单酶切验证？就是跑个胶看下大小？和pKD46差不多大小的plasmid多了
去了
做个多酶切 先看下plasmid对不对吧
E. |
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H*******i
发帖数: 196 | 9 “试过E.coli 另外的菌株BW25113也是出现同样问题”
质粒问题呗。 当然最好看看你们的培养箱的30°确认下没问题，不能确认就放室温
（25°）长，也应该能长出来。
另外多说一句，这个pKD46构建比较早了，KO效率不如一些新构建的。
还有听一些人说过BL21 用这个方法KO好像很难？ 不过这个我没验证过。 |
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