Genecloning three types of molecules? - (Oct/08/2011 )
What I understand from the original question is that you have already cloned the insert, and so you already have the recombinant plasmid. Any uncut plasmid (recombinant or not) would give several bands on a gel.
However, if you are testing the result of the ligation by analysing your colonies, you can have these results:
- Plasmid containing the desired insert. (Congratulations!). Recombinant plasmid, for sure.
- Religated plasmid (even if you used different restriction enzymes, religated products do sometimes appear, although if you treat with a phosphatase after digestion, this should be avoided).
- Original plasmid: because of incomplete digestion and religation (or no digestion at all) and you purified it together with the digested plasmid.
Since we generally clone into plasmids that have already been engineered for this purpose (think about multicloning sites, promoters, antibiotic resistance genes, etc) I would say all of them are recombinant.
To bob1: Yes, it looks very much like a homework question...!! perhaps I should not have answered.... but it will be his/her task to apply his criterium and believe what we have said, or not. We can always be wrong! ;-)
OA17 on Tue Oct 11 13:29:52 2011 said:
What I understand from the original question is that you have already cloned the insert, and so you already have the recombinant plasmid. Any uncut plasmid (recombinant or not) would give several bands on a gel.
However, if you are testing the result of the ligation by analysing your colonies, you can have these results:
- Plasmid containing the desired insert. (Congratulations!). Recombinant plasmid, for sure.
- Religated plasmid (even if you used different restriction enzymes, religated products do sometimes appear, although if you treat with a phosphatase after digestion, this should be avoided).
- Original plasmid: because of incomplete digestion and religation (or no digestion at all) and you purified it together with the digested plasmid.
Since we generally clone into plasmids that have already been engineered for this purpose (think about multicloning sites, promoters, antibiotic resistance genes, etc) I would say all of them are recombinant.
To bob1: Yes, it looks very much like a homework question...!! perhaps I should not have answered.... but it will be his/her task to apply his criterium and believe what we have said, or not. We can always be wrong! ;-)
Ah yes, I didnt concider the fact that most of those plasmids are allready altered for our use.