g*********5
发帖数: 2533 | 1 谢谢大家。看到thermo 有现成的敲除的strain卖，就是基因型看得头昏。
Strain Background Genotype
Mat A BY4730 MATa leu2Δ0 met15Δ0 ura3Δ0
Mat Alpha BY4739 MATalpha leu2Δ0 lys2Δ0 ura3Δ0
Mat A BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
Mat Alpha BY4742 MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
Het/Hom Dip BY4743 4741/4742
我要的菌株是Yeast HetDip Knock Out Strain YJR045C
怎么样的平板才能使敲除的菌株不长啊？能买到板吗？ |
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h**********r
发帖数: 671 | 2 做一个基因的knockout,加两端同源臂到loxp-ura3-loxp，转化，挑克隆，提DNA，用
PCR鉴定。问题是上游片段能扩增出来（一个引物在5' flank用于做construct之外,反
向引物在ura3之内），然后下游片段（一个引物在3' flank用于做construct之外,正向
引物在ura3之内）怎么都不work。试了好几个克隆，也换了primer.别的基因knockout
都没问题。按道理上游能扩出来，就表明基因被knockout了。想double check,结果下
游就死活不work.
请教大家可能的原因。多谢！ |
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h**********r
发帖数: 671 | 3 因为ura3和要knockout的基因size差不多，所以不太好比。准备把ura3去掉之后，直接
比size. |
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l**********1
发帖数: 5204 | 4 pRS416 excepts T7 also has Plac promoter.
pls refer
PMID 16820502
http://www.ncbi.nlm.nih.gov/pubmed/16820502
>pRS416 is a centromeric (CEN) plasmid and therefore is maintained at 1 to 2
copies per cell, while pRS426 is a 2μm plasmid, maintained at about 25
copies per cell (36). Both plasmids have the ColE1-derived pBluescript high-
copy origin of replication, a lacZα-associated multicloning site, an
ampicillin resistance gene (bla) for gram-negative bacteria, and a URA3
marker for yeast. These... 阅读全帖 |
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