cloning of long viral fragment I - (Jan/10/2005 )

Hi everybody,

I'm trying to clone a 8 kb viral cDNA fragment into a 3 kb vector. I tried it now several times and I'm really desperate because it doesn't work.

I get the fragment by pcr and through the pcr there are 2 restriction sites on either end of the fragment created. The restriction sites are BsiWI and NotI. There are 5 bp after each site to have a better cleavage.

After pcr (which is quite successful) I check it on a gel to see if it was successful. Then I do pcr purification with the qiaquick Pcr purification kit.
I check the amount again on a gel and estimate how much to use for restriction. I cut about 1 µg. First with NotI (over night) then the next day in the same buffer (is the same for both enzymes, NEB) with BsiWI. I do the same with the palsmid. I dephosphorylate the plasmid with Antarctic Phophatase (NEB, very new product but it works very, very good) and then do a gel extraction. The cut fragment I clean with a Qiaquick Spin cloumn. Then I check vector-Insert relation on gel. I then ligate over night , 16°C.

Ummmm...ya know you can digest with both enzymes at the same time if they use the same buffer. The first overnight digestion can be using up the buffer's goodies. If you use the same buffer without adding anything fresh the next enzyme might not be a happy camper...leading to poor digestion of the BsiWI ends. Unless you do add fresh buffer and I misunderstood your posting, then nevermind.....

Oh well...Either a double digestion or adding fresh buffer may be worth a shot. I hope it helps

I rarely do overnight digestions for ligation purposes. Here's why:

BsiWI
Ligation and Re-cutting:
After a 2-fold overdigestion with BsiW I, > 95% of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM) at 16ºC. Of these ligated fragments, > 95% can be recut with BsiW I.

Not I
Ligation and Re-cutting:
After a 10-fold overdigestion with Not I, > 95% of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM) at 16ºC. Of these ligated fragments, > 95% can be recut with Not I.

overdigestion relates to both enzyme quantity and incubation time. In the case of BsiWI, 2-fold overdigestion means you've lost 5% of your insert. Same reasoning for Not I.

Although theoretically, you only need a few vector+insert successful ligations, it does affect your ligation efficiency.