Cloning into pBluescript (pBS)

Could someone please help me?

What is the largest DNA insert that can be cloned into the Strategene's phagemid vector pBluescript?

I have amplified a 21 kb fragment by PCR and I need to clone into some vector

Thanks
ssiddara

Sounds to me like you need think about using a PAC (P1 artificial chromosome).

plasmids contain P1 origins of replication

(lysogenic & lytic)

advantages: stable as plasmid vector

lytic growth produces large amounts DNA

(but no phage particles)

easy to work with

clone & transform cells as plasmid vector

disadvantages: no size selection from packaging

do you just want lots of copies of the fragment or is there something in there you need to express in cells?

The longest insert I introduced into bluescript was 9 kb, but I've seen papers in which they cloned >20 kb fragments into the vector, so I guess it's possible to do it. In any case, I would advise you not to use normal DH5a competent cells for these types of long constructs, but some of those "ultra-competent" cells for transformation of large plasmids.
Cheers!

Cosmid vectors are great plasmids for an intermediate insert size (20-45kb) when working in e.coli. They work like regular plasmids, but are very efficient in maintaining the larger inserts. They were popular back in the day with 'finishing' to fill sequence gaps for genomic seqencing gaps. Unfortunately, I can't provide any specific cosmids for your purposes...

Pls dun use pBS... your insert is way too big.

If you are up to it... try using EMBL3/4 vectors... these are lambda phage vectors.

Otherwise, cosmids would be the next choice.