Cloning problems

Shanthi Ganeshan shanthi at casbah.acns.nwu.edu
Wed Apr 26 11:42:12 EST 1995


in response to not getting good results with the TA cloning kit, I would   
not necessarily give up. One of the critical steps in TA cloning is   
winding up with a A on the 3' end of your insert. Tag does not do this   
uniformly with all bases. According to Stratagene,(one of their flyers   
they put out-aprroximately 1 year ago), Tag puts an extra base on the 3'   
end of the polymerase product 50% of the time (the good news). 
Now for the bad news. Tag puts an A on the 3' end of the pcr product in   
the following order when the last base it reads off the template is: 
G: G>A>C 
C: C>A 
T: tag removes the T and puts on an A (resulting in a insert without an   
extra base on the 3'end of the insert. 
A: Tag puts on A on 50% of the time. 
 
So the secret of good TA cloning is to design primers with a T on the %'   
end of the primer-this obviously winds up with an A as the last base Tag   
polymerase puts on the insert before it puts on the extra base. 
 
Another important item is to use the highest competency level of cells   
that you can get. The cells that Invitrogen supplies with the kit are at   
best 1 microgram of dna gives 10 to the 8th number of colonies. I suggest   
you make your own cells to a competency level of 10 to the 9th or just   
buy them from stratene. The reason it is important to use really   
efficient competent cells is that TA cloning is not nearly as efficient   
as the standard sticky end cloning, although it is probably about 10   
times as efficient as blunt end cloning. 
 
The next important item is screen efficiently with  high throughput. Use   
the slot lysis method if your insert is at least 500 bases.  
 
start an overnight culture: 
 
next day take 0.5 ml and make your frozen stock, or store the culture at   
4 degrees until you figure out which cultures you want to make stocks out   
of after you do the slot lysis. 
 
take 100 microliters out for the slot lysis, and add 100 microliters of   
phenol/chloroform, vortex 1 minute, centrifuge in a microfuge at top   
speed for 2-5 minutes and load 30-40 microliters of the top layer on a   
gel (usually0.8 to 1%) in TBE. 
 
It is usually good to run a culture through the system that you know does   
have your vector plasmid with no insert. You are obviously looking for a   
slower moving band than your plasmid. You will see a very slow moving   
band at the very top of the gel (bacterial chromosomal dna) and probably   
two faster moving bands (ribosomal rna). 
 
Prep the remaining portions of the culture for dna if they have plasmids   
with the insert that you think has the appropriate size. I can do about   
100 cultures in 3 hours (slot lysis procedure). 
 
Let me know is this helps>




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