5 replies to this topic

[proteinz]

Hi All,
I have a query regarding construction of a phage library. I have purchased the Zap express vector kit predigested with EcoRI. From the literature other scientists digest with EcoRI (obviously) and perform the ligation normally. while others partially digest with 4bp cutters such as Sau3A1 and fill in two of the nucleotides. Why do they do this? Does it just make it easier to ligate or is there something i'm missing? Which is best??
Thnks in advance

[NemomeN007]

Difference in average insert length depending on the genome of interest.

[proteinz]

NemomeN007, on 16 August 2010 - 09:56 AM, said:

Difference in average insert length depending on the genome of interest.

Thanks for the reply nemomeN007 But i dont quite get this could you explain further? Why do they need to fill in nucleotides on 4bp cutter and not on the 6bp??

[NemomeN007]

It depends on the protocol and what you need to accomplish.  Without the protocol, I don't really know...usually it's to create a blunt end for linker ligations or variations thereof....

[proteinz]

NemomeN007, on 17 August 2010 - 05:15 AM, said:

It depends on the protocol and what you need to accomplish.  Without the protocol, I don't really know...usually it's to create a blunt end for linker ligations or variations thereof....

Thanks again for the reply nemomen007. the protocol is the ligation of restricted gDNA fragments into a lambda vector. In most protocols they cut directly with EcoRI and ligate into a vector digested in the same fashion. in some however the digest with a 4bp cutter such as Sau3AI then fill in 2 of the 4 nucleotides before ligating into the EcoRI digested vector. I understand the concept that 4bp cutter cuts more often and hence gives smaller fragments just not why they fill in two of the nucleotides before ligation with the vector.

[NemomeN007]

Again, I don't really know without seeing the protocol...