single restriction enzyme digestion? - is it possible to insert PCR product? (Jun/07/2006 )
depends, in my eyes site directed would be worth to think about, especially, if you want to use the then new created restriction site for more than one experiment. with the system we use in our lab here, it take approx 4 days to have the base changes made. this includes sequencing, which takes 2 days. the hand-on time is some hours.
1. PCR (few hours depending on cycler)
2. DpnI dig (1 hour)
3. transformation, o/n incubation (o/n)
4. pick colonies, mini prep (o/n)
5. Mini prep -> to sequencing
6. 2 days later you should have the sequence (or you use o/n service that some suppliers offer)
the screening can be more costly, if you have to sequence to validate. ligation into one restriction site need not to be 50/50 always. you should dephosphorylated the vector in this case for sure, otherwise you'll have mainly re-ligations.
So i would conclude, if you want to use this way of cloning for different inserts, i would go for site directed mutagenesis. if not, screening some colonies could be a way.
Kersten
Kresten, you mean site directed mutagenesis of the vector....i think it may be possible but time consuming no? i never did site directed mutagenesis.....so i might leave it as last option if others dont work....thanx for the idea!
Fred, ill think about restriction enzyme check....you are right i just can get 50%.....
you mean PCR check of the colonies right?
Just use a little bit of bacterial from clone on plate for PCR checking before you decide culture baterial clone and plasmid purification for sequencing.
PCR is less expensive than sequencing, I think.
yes i think i might use this for only one experiment.....
thankx a lot everyone!
Just double checking, so all I have to do is dephosphorilate the vector right? other steps should be the same as digestion with two REs.... 
Hi Kathy,
I am new to this forum. I have some experience with ligation of vectors and inserts that have the same sticky ends. What I did (taught by my supervisor) was that I digest the recombinant plasmids with two restriction enzymes. One of them cut inside the insert and other one cut on the vector. I need to find sites which are not at the centre of the insert. Then, by checking the length of the cut out fragment, you will be able to tell the direction of the insert. This method does not require PCR or site-directed mutagenesis.
If you are interested in this method and have any question, please let me know.
Cheers,
Charles
thanx a lot...i have thought of this before and yes there is such a site inside my insert....so ill be able to do this....but what you mean is that i dont need to dephosphorylate?
hi kathy,
i agree with charles. just pick 5-10 clones, make a plasmid-prep and cut with two different enzymes. one just before your insert and one inside your insert (not the centre) and than checked the lenght of the cutted fragments on an agarose gel. then you can calculate which clone is the right one. that's really a very simple method and i've done it several times before. as fred said direction of the insert is 50:50 one of the clones will be the right one.
good luck!
i agree with charles. just pick 5-10 clones, make a plasmid-prep and cut with two different enzymes. one just before your insert and one inside your insert (not the centre) and than checked the lenght of the cutted fragments on an agarose gel. then you can calculate which clone is the right one. that's really a very simple method and i've done it several times before. as fred said direction of the insert is 50:50 one of the clones will be the right one.
good luck!
so i will check the colonies by PCR using just forward primer and the method that uv described using XhoI.....but does it mean i dont need to dephosphorylate or it is another story?
.....i should dephosphorylate to avoid self-ligation since im using single digestion... please clarify this point to me...