Since InFusion now got mentioned, I cant resist, but give my naive non-
expert overview for cloning from the experience in my lab:
1. Typical sub-cloning: Add restriction sites in your primers, make
sure you have a few additional 5' bases overhang else the enzymes will
not cut, digest then your PCR product and put it in your expression
vector. Simply works if you do it nicely, needs restriction enzymes,
make sure these do not cut your gene of interest, ligase, etc.
2. Go through (TOPO)-TA cloning: the main advantage is that ligation
especially in TOPO vectors works really well, you do not need to add
to your oligos the overhangs but just the restriction sites, but then
you need to mini-prep the first ('transfer')vector, cut out your
insert again with enzymes, and put it in your expression vector. No
expression-ready vectors for TA cloning I am afraid, plus you need to
buy the vectors and they are not too cheap for a lot of cloning. In
general, I had found it easier to use, since many times I would not
get colonies when restriction cloning directly to the expression
vector, and it was my personal favorite for a while ...
3. Gateway cloning: Based on specific vectors and enzymes and a
recombination reaction. Although it was big hype, and its certainly a
big success in many areas, in Structural Biology it never delivered,
in my view. The main reason is that vectors were somehow not great for
expression and very often you would get unwanted sequences etc etc.
Need to buy vectors, enzymes, not cheapest.
4. LIC, Ligation independent cloning. It requires that you modify your
favorite vector to be compatible. No need for restriction digest or
ligation (thus it is as cheap as it gets!), no need to design every
oligo differently or think of restrictions sites, no special cells,
just needs a clean PCR product and you can have next morning the
expression cells ready. We use it and like it in my lab and many labs
I know, including quite a few SG projects. A protocol is available
from the CCP4 Community wiki:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/LIC_cloning
If you wish to try it, a His-3C and a no-tag vector for E.coli
expression can be made available from Vangelis Christodoulou,
e.christodoulou.AT.nki.nl , who created these specific vectors and
established the protocol for use in my lab. We have made over the
years about 1,000 constructs many of which yield soluble protein.
Other vectors are also available commercially or from academic
developers.
5. SLIC, Sequence and Ligation independent Cloning. The trick here is
that you do not need to modify your vector first like in LIC, you can
do SLIC in any vector. You need slightly longer oligos than LIC, but
thats fine. People that use it seem to like it, although there are
reports that it can be painful to set up, I hear a lot of positive
things. A protocol is also available from the CCP4 Community wiki
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/The_Milan_protocol_
...
which was contributed my Marina Mapelli, but you need to read a bit
more for how to design the long overhangs since
that issue is not covered in this protocol. To my understanding In-
fusion cloning is basically SLIC.
If LIC was not working well in our lab and we would not had had
vectors we like, SLIC could seem like the way to go.
Apologies if the list is incomplete, and I can admit clear bias
towards my experience. Its only intended on the hope it can be
genuinely useful.
BTW, we (well, Wijnand Mooij in my lab) has developed a very basic
tool to design your cloning oligos for whichever method. The
innovative thing in it (although I am sure there are other tools like
this) is that you input the DNA sequence for your protein, then it
translates it and interrogates a handful of Secondary Structure
Prediction services, Disorder Prediction Services, domain linker id
servers, domain assignment servers, coiled coil servers, and displays
a simple output below your protein sequence. You can then click at the
domain borders you prefer based on this 'condensed' analysis view, to
select possible N- and C-termini for deletion constructs, and click
one button to display all the possible constructs that can be made
with these termini, and the oligos that you need to order for PCR. You
can add to these oligos your favorite overhangs (the default ones are
for one of our His-3C-LIC vector). You are welcome to use it at:
http://xtal.nki.nl/ccd/
ccd: Crystallographic Construct Design
and provide any comments or input. Apologies but there is no help or
documentation, but hopefully its dead simple to use.
Tassos
On Sep 1, 2008, at 16:44, Brian Wengerter wrote:
Alternatively, you could skip troubleshooting digestion/ligation/
etc. and use a kit based on site-specific recombination, like the In-
Fusion kit that Clontech sells. (I don't have any financial
interest here--I'm just a graduate student, but I've had good
results using it.) The kit is not without its own downsides--it's a
bit pricier than traditional cloning, you'd have to design another
set of specific primers, and you have to be very careful about the
vector:insert ratios you use.
Good luck,
Brian
Artem Evdokimov wrote:
Hi,
First of all – I am curious why did you decide to put in an extra
step (the T/A cloning into an intermediate vector)? You can
happily digest your PCR product with NheI/BamHI, clean up and
ligate into the appropriately digested pET-23a(+). If you have
issues, you should definitely try this.
Now, since you do have an intermediate step – did you verify that
everything was OK after havig subcloned your insert into whatever
vector you’re using? Did you sequence the insert and most
importantly did the sequencing confirm the nature of the linker
regions?
The enzyme pair that you chose has a slight issue with digestion
buffer – most people would choose NEB buffer 2 (since buffer 3 is
bad for Nhe) where Bam still has ‘100% activity’ – however, in
buffer 2 you can have star activity of the Bam due to the somewhat
lower salt concentration (50 mM instead of the optimum 100 mM).
It’s not impossible to imagine that you have issues with digestion.
This can be easily avoided by sequential digestion although of
course it’s slightly more work (but if you cut out the T/A cloning
step that’s actually still faster).
So, in conclusion the most likely issue is digesiton (probably of
the pET vector, to be more specific). Next likely issue could be
ligation – make sure that you base your ligation ratio on the gel
intensity of the bands as well as on the OD260 of your DNA. Faulty
primers are not likely to be an issue since you seem to be able to
restrict your insert out of the intermediate vector.
Please note that you can often use SpeI or XbaI instead of Nhe
since they have compatible sticky ends. Clearly this depends on the
vector you’re working with and I am too lazy to look up pET23
polylinker.
Artem
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf
Of vijay srivastava
Sent: Monday, September 01, 2008 3:06 AM
To: [email protected]
Subject: [ccp4bb] regarding cloning
Hi,
I am trying to clone a 1.2kb insert into a expression vector pET
23a through T/A cloning. The restriction enzyme used is Nhe1(NEB)
and BamH1 (NEB) in the forward and reverse primer recpectively. I
was succesful in subcloning (T/A vector) and getting my insert at
1.2kb after double digestion and also the vector at 3.7kb ,for the
ligation i am using the ratio of vector to insert is
1:3,1:2,getting the colony after the transformation but some how
when i used to confirm my clone through double digestion i am not
getting my insert at the correct position.Some time in the gel only
the size of the vector was there.
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