3 bands after digestion - (Sep/12/2006 )

hi

i was using a nrew endonuclaese and after the digestion it gave me 3 bands !!! isnt right that enzymes give u on agarose gel only one or 2 bands coz they cut the known sequence in the double stranded pcr product ??????????

---

If you were cutting a plasmid, three bands could indicate a complete lack of cutting, and thus you're seeing three different forms of the plasmid -- open circle (nicked), linear, and supercoiled...

---

I don't quite understand what you are saying. So correct me if my assumptions are wrong.
The restriction enzyme in use, cuts your plasmid in two places, so you should see 2 bands.

If that is the case, aside from Homebrew suggestion of total failure of cutting. Incomplete cutting would lead to three bands

Let say your plasmid is 2000bp, and cuts in two fragment 800, 1200

A single cut - 2000bp band
Double cut - 1200bp and 800bp bands

In total you see 3 bands.

P.S : As you were not clear quite clear in your post, it must also be realised that a restriction enzyme can cut a plasmid more then twice, 3, 4, ....17 times.

---

ITS NOT A PLASMID ITS A PCR PRODUCT FOR A aDNA

---

what if ur PCR had sites in between the gene which might have been overlooked.

---

My apologies. Still, my explination remains applicable. If your digestion is incomplete, some of your products will be cleaved in half, while the rest remain uncut. This would result in 3 bands, an uncut PCR fragment, and 2 smaller products from the digest.

---

Do you know how many recognition sites for your enzyme your PCR product has? (you don't seem sure; you say 1 or 2 bands expected but not 3. 1 or 2 bands from a PCR product suggests either no recognition site or one recognition site assuming digestion is complete. Which one???).

If you know where the cut sites are you can then determine the size of the expected products.

---

Did you gel purify your PCR product before digesting it? What does an uncut sample of your PCR product look like? What band sizes did you get on the digested sample, and what fragment sizes were you expecting? How big is your PCR product?

We can probably help you figure this out, but we need more information (nowhere in your first post, for example, did it say you were cutting a PCR product).

---

hi

thanks everybody for trying to help me and sorry for not beig so clear in my question....
im working on a PCR product of human DNA on a polymorphism ...the PCR product size should be 310 bp ( according to the paper im implying ) and it should cut after digestion to 2 bands (180 and 130 ) while we can also get 310 bp (uncut) or 310 +180 + 130!!!!!!

i dont understansd how can we get the uncut band toghether with the 2 cut bands after digestion in the same lane ???? (getting 3 bands after digestion (how??)

---

That's because part of your PCR product is cut by the enzyme and part is not cut. Try to add the enzyme, perform the digestion for th edesired time, add the enzyme again and digest. Be careful you don't add too much glycerol anyway!

---