>>today I have a really serious question t everyone beeing familiar with
>Here my problem:
>>Since more than a century now, I am trying to clone a PCR fragment, which
>I designed XhoI-XhoI, into an Xho-NheI. However, since today I have not
>been able to get a single positive clone. Why?
Wow. You have been doing this a long time 8-)
>Does anyone has valuable suggestions, I really would appreciate it.
>Thanks in advance.
I believe some of your problem may be related to XhoI and NheI not having
compatible overhangs - XhoI gives a TCGA, while NheI has a CTAG overhang.
You could either:
a) try using an enzyme that has an overhang compatible with XhoI, such as
SalI (I dont know of any others offhand.);
b) redo the PCR using primers with the NheI site instead of the XhoI site.
This will of course take a bit longer, since you will have to get the
primers and do the PCR, but if the vector you are cloning into does not
have a SalI site in a place you can use, it may be the only way out for
c) look in your sequence for other restriction enzyme sites tht would be
more useful for your application.
megan at ucla.edu