i'm trying to clone my fragment of 255bp with nde1 and xho1 restrictions enzymes but still I don't get the fragment. i used 1:3 ratio,1:ratio,1:6 ratio but I always get only the recombinant vector, the enzymes work well.as my fragment is small I've tried digestion for 3h and 4h but nothing. can I digest it for overnight?
Hi
I would rather assume that you are digesting too long. The number of sequences to digest is not related with the length of the insert. Please check if your restriction sites are in direct proximity to the 3'/5' ends of your insert. This may affect the restriction efficiency
Sven
Sven Reister I think so because the primers I use contain the restriction site.5'gcatcatatg....template, the 3'end also has xho1 restriction site. i'm repeating this cloning for ninth time but still nothing.
Hi
Pls consult the NEB homepage. There is a list for restriction efficiency in proximity to the sequence ends. I have always added 4 to 5 bases between the end of the recognitiin site and the end of the sequence
thankyou Sven Reister ; can you tell me how long time did you digest in that case?as i know ndeI take time to recognize four bases at the end; but my case is four bases between the end of restriction site and the end of sequence: Can someone advice me how to do this digestion?please i need help; share your experience
HI
Here is the link. It clearly shows that NdeI needs some bases as stiffer at the end. Would suggest to redesign the primers and perform digest as recommended in the enzyme handbook
Sven
https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
I think digestion is not the problem here. Your primer does have 4 nt before the NdeI site (and I presume you have similar spacer for the XhoI site). It is more likely the problem is something else, such as your purification of the insert, or the ligation reaction itself, or the transformation, or perhaps the vector isn't completely digested. Try running some controls, such as unlighted vector alone (what is the background) and ligated vector without insert.
Unless you use way too much enzyme, digesting overnight should not be a problem with NdeI or XhoI. We do this all the time.
However, you should gel isolate your NdeI-XhoI fragment for further cloning.
As for the vector, besides cutting with NdeI-XhoI, I would also recommend digesting the cut-out using a blunt-end cutter (guard against re-ligation), as well as treating with CIP/SIP to dephosphorylate the ends. I would also recommend gel isolating the vector fragment as well to remove any un-cut vector.
Good luck
Randolph B Caldwell do you mean I should run the gel for fragment digested? i u sually do the purification directly after fragment digestion
I am suggesting that you should gel isolate both your digested fragment and digested/CIP treated vector instead of doing a only a column purification or alcohol precipitation.
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