基于Li-COR的southern blot,求帮助# Biology - 生物学
R*n
1 楼
最近做一个genomic feature需要用20bp左右的probe hybridize digested genomic
DNA.有几个问题
1）overnight transfer DNA 到 nitro membrane之后，压扁的胶里还是有 DNA smear,
是不是没有transfer成功，还是有残留？
2）LI-COR的protocol是对PCR probe优化的 （65C hydridization, 50C wash），
https://app.boxenterprise.net/s/swc2yyeseopg608jlyiuqe8qs9t33rb9
怎么优化hybridization温度？这篇文章提了一下.关键这个hybrization溶液里有
Dextron Sulfate 和 近 700mM的Na+.算算有68C了。。。
http://www.alliot.fr/BIO/PDF/SouthernBlot-.pdf
short oligonucleotide probe is used (15–25 nucleotides in
length),then the hybridization step is usually carried out
under conditions of low stringency (typically at a
temperature several degrees below the calculated Tm for
the desired hybrid),so that all potential hybrids,including
nonspecific ones,are able to form。 Specificity is then
achieved by a series of washes at increasing temperatures so
that,hopefully,only the desired hybrid remains at the end
of the procedure.
3）probe是biotin labelled.我看了一下以前的讨论，牛奶肯定不能用来bloxk。 1%
BSA 应该没问题吧？
DNA.有几个问题
1）overnight transfer DNA 到 nitro membrane之后，压扁的胶里还是有 DNA smear,
是不是没有transfer成功，还是有残留？
2）LI-COR的protocol是对PCR probe优化的 （65C hydridization, 50C wash），
https://app.boxenterprise.net/s/swc2yyeseopg608jlyiuqe8qs9t33rb9
怎么优化hybridization温度？这篇文章提了一下.关键这个hybrization溶液里有
Dextron Sulfate 和 近 700mM的Na+.算算有68C了。。。
http://www.alliot.fr/BIO/PDF/SouthernBlot-.pdf
short oligonucleotide probe is used (15–25 nucleotides in
length),then the hybridization step is usually carried out
under conditions of low stringency (typically at a
temperature several degrees below the calculated Tm for
the desired hybrid),so that all potential hybrids,including
nonspecific ones,are able to form。 Specificity is then
achieved by a series of washes at increasing temperatures so
that,hopefully,only the desired hybrid remains at the end
of the procedure.
3）probe是biotin labelled.我看了一下以前的讨论，牛奶肯定不能用来bloxk。 1%
BSA 应该没问题吧？