AscI digestion of plasmid - Problem with AscI digestion of plasmid DNA (Nov/08/2006 )

Dear All,
I am struck with the digestion of 6381 bp long plasmid with the AscI and PshAI. Both the enzymes are of NEB and I had done double digestion with both of these. I had added 1ul of each enzymes in reaction volume of 30ul including 1.5ul DNA. Digested at 37C and also 25C as per the NEB. But I am unable to see the ~300 bp product. The heavy band is seen but it is very difficult to identify if it. And the ~300bp band is not seen.
My question is that does anybody had used AscI of NEB previously? What are the main things to take care? Is it working well?
Thanks in advance!

Hi Niraj,

NEB says that AscI is strongly inhibited by NaCl and ammonium acetate. They recommend using sodium acetate or potassium acetate as the salt for alcohol precipitation of DNA to be cut with AscI. And because of this it's very important to use buffer 4 for AscI.

Btw, PshAI needs BSA and should be used at 25°C if you digest longer than one hour.

Maybe you should check both enzymes seperatly if they are cutting the plasmid. And maybe they are not working well together. So maybe a two step restriction will help?

That's all I could suggest.


Chakchel

Hi Chakchel,
Thanks a lot for your reply. I had done the digestion of other plasmids with PshAI and AscI and they are working. I doubt about the efficiency of both the enzymes in working together.
I had eluted the visible band and had performed the ligation with 36 bp insert.
The results were like this on LBA with Amp.
I [vector+insert+ligase]: 3 colonies
II [vestor+ligase]: 35 colonies
III [vector+insert]: 0 colonies.
Please anybody or u can explain why this happened?
Hope to hear you soon.
With best regards,
Niraj
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Hi Niraj,

hmm, to me your results sound like most of vector is only cut once, so you get lots of religated vectors in II. The ligation only works with this one end of your vector so in I. the "half ligated" vector is unable to close, so you get less colonies.

Did you check those three colonies of I. already? When your insert is in there you are lucky and had some double cutted vector.

To be sure that only once cutted vectors won't religate I would suggest you to dephosphorylate the vector after digestion.

But you should try to find out which one of your enzymes didn't cut properly by making one digestion for every enzyme with this vector... even when they usually work well.

Hope it'll help! Good luck!

Chakchel

Hi Chakchel,
Finally I got the results and 11/20 were clones. The 3 coloines of I were negative.
I am happy now!!!
Thanks for you interest and suggestions!!!
Niraj

Hi Niraj!
Congratulations! That's great! Did you change anything?

Greetings,
Chakchel

Hi Chakchel,
What we did is we ran our digested products on the 1.2% (instead 0.9% our lab norm).
Thanks all!!!