Subcloning dirty PCR products
sswift at umbc.edu
Thu Aug 25 18:11:09 EST 1994
Emma Macfarlane (emma at HERA.MED.UTORONTO.CA) wrote:
: Hi German
: You didn't mention what restriction enzymes you are using. Have you checked
: to see if they will cut close to the ends of linear DNA? I recently spent
: a long time trying to subclone a PCR product which I thought I had digested
: with Nde I and Xba I only to find Nde I needs a minimum of 7 base pairs after te recognition site to cut efficiently (the appendices in the NEB catalogue are
: good for checking this kind of thing) and I had designed my primers to give
: only 5.
: Hope this is some help. Good luck
I have done a lot of these for my research. The enzymes I used were BamHI,
EcoRI, XhoI, SalI. On each of these I only had 4 bases flanking the RE site.
What I used to do was PCR the fragments, phenol/chloroform( not recommended by
most, but it worked for me), EtOH ppt., then resuspend right into the desired
RE digestion mix. The digestion would be using 10 to 40 units RE per PCR sample
and done for at least six hours. Usually I would either digest at 37C overnight
or the digest would be for ~4 hours at room temperature, then followed by the
37C overnight incubation. The reasoning behind the Room Temp digestions was
that this would cut back on the breathing of the ends of the PCR products and
this would increase RE digestion. Also it helps to purify after digestion,
either by gel, Geneclean(or the equivalent), or even just by EtOH ppt, though
I have done with out cleaning after digestion. W/out cleaning I would heat
treat the digest mix at 70C for 15-20 minutes (even for Heat resistant REs),
and then store on ice or 4C. Then maintaining everything at temps below
16C, I would mix in all the ingredients for the ligation, and ligate at 16C,
for 6 hours minimum. The bacterial transformation was done using the ligation
mix as is.
In about a year I did about 25 PCR clonings. (not so good these days?)
Then again I had to learn it all on my own, without any internet available for
Someone mentioned the TA cloning system for PCR cloning. This is a great system
except that it requires you to subclone afterwards. In my case I had to screen
the clones for the PCRed gene's function, and I had to rePCR several because
mutations which killed the function of the gene product. Even so I have heard
that the TA system for cloning PCR products is very fast, and subcloning isnt
that hard. Getting the PCR product in to a vector the first time is hard.
Subcloning is a breeze in comparison since you can produce copious amounts of
DNA to do it with.
If the TA system is as fast as I've heard....
If I had had access to such to a TA vector when I made all my plasmids,
I probably would have used it, even with having to rescreen everything.
Steve - a wanderer lost on the Net.
Have disclamer, will travel.
"DON'T PANIC!" - D.A.
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