PCR CLONING POLL RESULTS-AT CLONING WINS!

[Morris F. Manolson]

Here are the PCR CLONING POLL RESULTS.  Out of 30 responses....

TA cloning:         14 votes
restriction sites:   9 votes
Blunt end ligations: 6 votes
others:              1 vote  (CloneAmp kit from GIBCO-BRL)

There were 4 votes for InVitrogen's new PCR kit
(but everyone complained about the cost), three votes
for Novagen's pT7blue vector which is cheaper as they
just sell you the vector, and several methods on how to 
make your own vector for AT cloning.  I have enclosed all the
comments on the bottem of this post.  Thanks again to everyone
who has responded!  This has been GREAT!

I have tried InVitrogen's PCR1000 a long time ago without success. 

TA cloning gets my vote. You may also have been refering to my net about
the vector we use, pDK101. It works very well in most hands, and has always
worked for me even if it took a few attempts. You can read more about it
in Kovalic, D. et al. 1991 NAR 19:4560-4563.

We like the TA cloning method.  I haven't been unable to clone
a PCR product yet using these vectors.  I like PCR2000 (?)
from Invitrogen because it has both SP6 and T7 primers for
DNA sequence analysis and a polylinker that is very convenient
for further subcloning.  We're now using Novagen's pT7Blue.
(Because it is cheaper - novagen allows you to buy just the
vector, not a whole kit!)  The polylinker isn't as good and it
has a different sequencing primer built in, but it works!
 
BTW, Invitrogen's first vector, pCR1000 was based on the
enzyme HphI, so you could engineer a vector yourself.  

	People here at the station are fond of another method. It's 
taken from a paper in Nucleic Acids Research: Vol 20, 1992, pg.6427.
You simply omit the 10 minutes extension at the end of the PCR 
reaction and use the reaction directly in a ligation with a blunt
cut vector (not dephosphorylated). Apparently it works well for 
small fragments (<1kb). I hope this is helpful. Les.


TA TA TA TA TA
before I knew about TA vector, I spent a month or two unsuccessfuly trying to
fill
the ends and blunt ligate my product. I finally ended up ordering new primers 
with cutting sites on the ends. (This was a few years ago)
My lab now is rather dependant on the invitrogen TA kits. (except for a bad
lot recently, it has ALWAYS worked really well for me.  We recently got a new
post doc who decided to try and save money and make vector.  She used (I think)
the red book method, which just cleaves your plasmid of choice at a blunt site,
then just put with Taq pol and dAtp for  a while, and bingo. Ta vector. Her 
first try was a success. Besides the cost savings, it solves one big problem:
the invitrogen vector has a REALLY long distance from the m13 primers to the 
TA site, so you have to run pretty long gels to even get to the insert which is
just at the XC dye (around 150 bp from the primer??). Using the red book
method,
you can have any vector you want!

I've tried lots of ways and all have worked. TA cloning with the invitrogen
kit gave me only one positive, and wasted two months of my time. Blunt
ending was my first technique for cloning V-ATPase a la Bernasconi, and
gave me a few dozen colonies. Generating overhangin Ts in blunt-cut
bluescript (using Taq and only dT present) to create home-made T-vecotrs
has worked well with a friend. But now, I spend the extra $10 and put a
site at the 5' end of each primer. It always works, and by using Eco and
Bam sites I can cut in a single reaction buffer, then directionally clone.

T-vector, using blunt-cut pSK or Bluescript T-ended using Taq and dTTP. 
Or blunting in straight, using Amersham blunt-end cloning kit.

	I tried using T-vectors (both homemade and Novagen's TBlue kit) and had
no luck at all.  I finally had success using the EcoRV site of pBSKII+ and
using T4 DNA Polymerase to exonuclease and (re)-fill in the PCR fragment to a
blunt end.  This is the method I would recommend.

We first tried using the restriction sites introduce by the primers but
got nowhere for months! Was it because one of them was Hind III?
      TA cloning (Invitrogen) was next and after some early loses it
was finally sucessful. 

I vote for 2).
Method: PCR, add 1 ul T4 pol, 37 deg for 10 min, clean DNA, ligate.

 
My vote goes with...
 
>       3) designing oligonucleotides with restriction sites on the 5' 
>          ends
 
Works every time; I just phenol/CHCl3 extract PCR products 1x, then put aqueous
phase straight into a G50 spin column.  Whatever comes through gets cut, then
precipitated with isopropanol, and it always seems to clone.

	My vote is for #4 and specifically the CloneAmp kit from
GIBCO-BRL. You synthesize the primers with repeats of CUA or CAU on the 5'
ends, do your PCR, take an aliquot of the reaction mixture and the BRL
cloning vector and treat with uracil glycosolase (SP) for 30 min,
transform, and voila-lots of colonies. It has been my experience that the
false positives for this method is low (<5%) and I have been able to clone
everything that I PCR.

I use TA clonning to clone PCR products into vector and ussually it is 
the vector from Invitrogen.

I "TA" clone with the Invitrogen kit.  It's expensive, but works very well
and save lots of my time.

Easily TA cloning. We use the pCRII vector kit from InVitrogen. I've had
PCR products with restriction sites at both ends and the efficiency was
still much better using the TA method rather than cutting first.

TA Cloning Kit from Invitrogen using the latest vector. Works extremely well
in my hands however the competent cells provided vary greatly in competence.
Also the use of a Amp plus Kan vector is a real pain when moving the PCR 
fragment into say an expression vector which is nearly always Amp or Kan
itself.
Just creates a bit more work isolating fragments etc. Novagen TA kit also works
but I found it gave a much poorer ratio of positive clones ie 10-20%
against at 
least 90% for TA.

	I've used methods 1 (TA cloning) and 3 (design a restriction site
into the primer).  Method 3 worked not at all for me, but that was my
fault--I didn't check to see that the enzyme would cut that close to the
end. (I was young.)  My T-vector was constructed by cutting Bluescript
with EcoRV and incubating with Taq and TTP, although any vector with a
site for a blunt-cutter could be used in its place.  The method is from a
paper by Douglas Marchuk, Mitchell Drumm, Ann Saulino, and Francis S.
Collins, which was faxed to someone who dated someone who worked in my lab
in February of '91, but I don't know where the paper was published.  


 
We find blunt-end cloning into a Sma cut vector in the presence of
Sma easy, reproducible, and very satisfactory (other blunt end cut
vectors can also be used). Polish the PCR product first with a bit of
Klenow.







******************************************************************************
Morris F. Manolson                     Tel:  416-813-6662  (office)
Division of Cell Biology                     416-813-5594  (lab)
Hospital for Sick Children                   416-813-5028  (FAX)
88 Elm St., McMaster building        email:  Morrie at resunix.ri.sickkids.on.ca
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