Dear Olanrewanju

i think that you can try a different approach based on vector amplification by pcr.

- you can add to the primers a New restriction enzime sites ( which of course dont have to be present into the vector) and digest the purified pcr to see if the problem is related to the specific enzyme efficiency

- you can add overlapping sequences that make this vector a V-PCR suitable for PIPE cloning.

https://www.ncbi.nlm.nih.gov/m/pubmed/18988020/

9.5 kb is s long pcr but with a good high fidelity polimerase i think that it is feasible.

if you are interested to know more detail on PIPE cloning, you can find on my blog: https://proteocool.blogspot.com/ ( ProteoCool n°1: cloning method overview and ProteoCool n°3 : Primer design for PIPE cloning) more infotmations about it

good luck

ciao

Manuele

Hi Oni,

I have the opinion that what you need to do first is more of a control check before trying other options.

1. The protocols for every commercially available restriction enzyme is stated by the manufacturer. Its always a straight forward instructions but you can always optimize the conditions especially time of incubation and amount of the enzyme/DNA sample used in a given reaction mix. Thus, confirm that you are following the correct instructions and your enzyme is still active, properly stored or shipped, and that you are using the correct digest buffer.

2. Confirm that you are using the correct MsAp1I restriction enzyme and sample (F88-4 phage vector), it is possible to get the wrong things in the rightly labelled tubes and vice versa . Hope you are using a purified sample and not a total DNA sample from E.coli? If not try a purified sample.

But for the records, MsAp1I restriction enzyme is blocked by CpG (methylation) overlapping when using a eukaryotic genomic DNA but should not have any effect on your system as you rightly said (https://www.neb.com/tools-and-resources/selection-charts/dam-dcm-and-cpg-methylation).

Hope this help. Let us know.

Cheers