Cloning Problem - (Nov/04/2006 )
Hi guys, again im facing a new problem. I am trying to clone a 260 bp insert into a 10kb vector using Sma1 (blunt) and BamH1 (sticky) . I first digested the insert and the vector using Sma1 and Buffer 4 (NEB) at 25C for 1 hour, then clean up. Then i digested it it with BamH1 for 1 hour at 37C using Buffer 2, followed by clean up then ligation O/N at 16C, using 1:3 and 1:6 ratio. i then electroporate it using competent E. coli cells and plated on Amp plates with X-Gal. Where do u think i made the mistake? bec i have no transformants 
Thank you for the comments
hmm.....
The vector is 10kb, meaning it would rather recircularise to itself then pick up any insert. Especially a blunt end (aleast on one end) ligation.
Is it at all posible to use a different stategy so that the small insert comes in earlier and ligated to a smaller version of your vector?
What is the volume of your ligation? It should be small and the DNA concentrated to overcome the above problem. I would have 300-500ng or so of DNA in 10ul. The ratio is looks okay... may want to keep to the 1:3 mol ratio.
How long did you ligate this for? I would go for an overnight ligation.
The next question is, have you dephosphorylated your vector? It was not mentioned.
SmaI is also not a quick enzyme... I would suggest you cut your insert longer then 1hr. I would go for an entire evening. BamHI is a fast cutter.
Do you know if your ligase and ligase buffer is fresh and working... This is always a problem.
The vector is 10kb, meaning it would rather recircularise to itself then pick up any insert. Especially a blunt end (aleast on one end) ligation.
Is it at all posible to use a different stategy so that the small insert comes in earlier and ligated to a smaller version of your vector?
What is the volume of your ligation? It should be small and the DNA concentrated to overcome the above problem. I would have 300-500ng or so of DNA in 10ul. The ratio is looks okay... may want to keep to the 1:3 mol ratio.
How long did you ligate this for? I would go for an overnight ligation.
The next question is, have you dephosphorylated your vector? It was not mentioned.
SmaI is also not a quick enzyme... I would suggest you cut your insert longer then 1hr. I would go for an entire evening. BamHI is a fast cutter.
Do you know if your ligase and ligase buffer is fresh and working... This is always a problem.
hi perneseblue, tnx for the reply. I used a 10 uL volume for the ligation. Overnight at 16C. But i guess i didnt reach that 300-500 ng DNA concn. Mine is approx 100 ng only. So thats a good tip perhaps, il do it once again using that DNA concn.
I didnt do any dephosphorylation.
Ok il take ur suggestion. Il do the digestion with Sma1 longer than 1 hour. il do it O/N
The ligase and its buffer are ok. I guess the above problems mentioned are the cause. thanks for your suggestions.
