ligation product- direct pcr - (Oct/06/2006 )
hai
i want to insert around 100-500 bp genomic restriction digested products into my vector (8 kb).
after ligation i am interested in amplifying a region of say around 7 kb. can i do the amplification directly aftr. ligation. there was a post in this forum where a person has done one like this and it was said that the ligated product was run on a gel and the gel slab was directly used as the template for pcr. can i know much more details abt. this?
please help me out with this. struggling with this as i get many shorter sized fragments in pcr.
-bioenthu-
QUOTE (bioenthu @ Oct 6 2006, 10:33 PM)
hai
i want to insert around 100-500 bp genomic restriction digested products into my vector (8 kb).
after ligation i am interested in amplifying a region of say around 7 kb. can i do the amplification directly aftr. ligation. there was a post in this forum where a person has done one like this and it was said that the ligated product was run on a gel and the gel slab was directly used as the template for pcr. can i know much more details abt. this?
please help me out with this. struggling with this as i get many shorter sized fragments in pcr.
i want to insert around 100-500 bp genomic restriction digested products into my vector (8 kb).
after ligation i am interested in amplifying a region of say around 7 kb. can i do the amplification directly aftr. ligation. there was a post in this forum where a person has done one like this and it was said that the ligated product was run on a gel and the gel slab was directly used as the template for pcr. can i know much more details abt. this?
please help me out with this. struggling with this as i get many shorter sized fragments in pcr.
HI
I am new here, is that really possible ?
up to me, even using Roche ligation kit, which have the highest ligation efficiency I ever known, can give only very small amount of ligation product, I have never using ligation product as template of PCR, but I just wonder, after purified by gel electrophoresis, the remained ligation product is really enough for PCR ? If it is possible, it too really helpful for me
-lactamase-
that was a topic for discussion in this forum some time back. i am also in search of that protocol.
those of u who know, please respond.
-bioenthu-