1 reply to this topic

[krystle]

Hi guys, i'm interested in cloning a 25kb fragment from genomic DNA. After reading up on it, i realise that i can do it via 2 way - either single long range pcr, or do multiple pcr.

My question is, which method is better?

From what i understand, for long range pcr, i'll need to get special pfu enzyme mix thats capable of pcr such long fragments, and there will be strict requirement on the quality of the genomic dna template used. I've been reading up on a few kits from qiagen, invitrogen,fermentas and etc but i'm really skeptical about it. Has anyone has success with any particular brand for pcr such long fragments?

As for the second method, i will have to pcr the 25kb in smaller fragments, so that i have maybe 6-7 fragments that are 3-4kb in size (which can be pcr out using standard protocol). However, that would mean that i will need to do multi way ligation. I've seen people doing 3 way ligation but if i follow this method it will mean that i need to do 7-8way ligation. Do people perform such ligation?


Please help me!  

[Warren]

krystle, on Sep 16 2009, 08:38 AM, said:

Hi guys, i'm interested in cloning a 25kb fragment from genomic DNA. After reading up on it, i realise that i can do it via 2 way - either single long range pcr, or do multiple pcr.

My question is, which method is better?

From what i understand, for long range pcr, i'll need to get special pfu enzyme mix thats capable of pcr such long fragments, and there will be strict requirement on the quality of the genomic dna template used. I've been reading up on a few kits from qiagen, invitrogen,fermentas and etc but i'm really skeptical about it. Has anyone has success with any particular brand for pcr such long fragments?

As for the second method, i will have to pcr the 25kb in smaller fragments, so that i have maybe 6-7 fragments that are 3-4kb in size (which can be pcr out using standard protocol). However, that would mean that i will need to do multi way ligation. I've seen people doing 3 way ligation but if i follow this method it will mean that i need to do 7-8way ligation. Do people perform such ligation?


Please help me!  

I suspect, that in the second method, what you do is not a 7 or 8 way ligation, but rather your first PCRs are designed to overlap so that you can mix the first PCR products, using only outside primers, and create a full length molecule via PCR.  If you knew convenient restriction sites in your genomic DNA, that would be a way of piecing it together as well via PCR of restriction fragments and subcloning, which is how I'd probably chose to do it if possible, but 25 kb is pretty huge (for PCR or regular plasmid cloning)!  Warren..