Megan megan at ucla.edu
Sun Sep 15 16:14:24 EST 1996

>Hi everybody,
>today I have  a really serious question t everyone beeing familiar with
>molecular biology.
>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
you, except;

c)  look in your sequence for other restriction enzyme sites tht would be
more useful for your application.

Good Luck!!!

megan at ucla.edu

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