Ligase free annealing question

I need to do the following:
1) mix and anneal 2 primers that after annealing should produce a 36bp
double-strand region and 15nt sticky ends on both sides. This product will
be used as an insert.
2) use above insert to anneal with the linearized plasmid having
complementary sticky ends to the sticky ends in the insert.

Does anybody have any suggestion on how to increase the yields of both
annealed inserts and efficiency of insert incorporation into the plasmid?

I know this works, but unfortunately I don't have a detailed protocol.
Important parameters like concentrations of oligos in the annealing
reaction, buffer and salt concentrations, temperatures, ratios of molecules,
etc. are missing.

Any reference or pointer would be highly appreciated.
Thanks.
Emir

.....two complementary oligonucleotides containing AR binding sites
were obtained from commercial provider (MWG AG BIOTECH, Germany). In
addition to AR binding site oligonucleotides contained two
5'-overhanging deoxyguanosines for fill-in labeling of resulting
oligonucleotide overhangs with radioactive alpha-[32P]dCTP using the
Klenow fragment.

Oligonucleotides AR-1 (10 microl) and AR-2 (10 microl) were mixed in
equimolar amounts (1 nmol each) in 70 microl of ddH2O and 10 microl of
10x annealing buffer (500 mM TrisHCl pH 8.0, 100 mM MgCl2 and 1M
NaCl). Annealing reaction was performed in PCR machine (5 min 95°C, 10
min 70°C, 10 min 65°C, 10 min 60°, 10 min 55C°, 10 min 40°C).
Resulting double-stranded oligonucleotide concentration was
approximately 200 ng/microl.

"redeamer" <[Only registered users see links. ]> wrote in message
news:a86fd891.0401230813.614821ab@posting.google.c om...

Thanks a lot! How about annealing to a linearised vector with complementary
sticky ends? Would same buffer and say 50-fold molar excess of the insert
vs. vector be OK? The problem I guess is in the efficiency, because annealed
vector/insert product will be used to electroporate E.coli, and if the yield
of closed circle is too low, no colonies will show up...
-Emir

Emir,

This is a very simple protocol that I use:

Mix the complementary oligos in equimolar concentration (TE, pH7.5 or 8 with
50mM NaCl) in a 1.5mL tube. I usually start with about 500picomoles of each.

Boil about 500-700 mL water in a 1L beaker, remove the beaker from heat source
(microwave or burner or hotplate), and float the tube of oligo mix on the hot
water (using a floatie, of course). Let the water cool to below 30 degrees.
When I do it, I simply let it sit on the benchtop overnight.

Alternatively, you can also do it in a thermocycler using the preset
"denaturing" (95 C) for 5min, and then programming the block to cool to 25 C
over at least an hour.

The anealed oligoes are stored frozen at -20. In some cases, such annealed
oligoes may need to be re-annealed before using after a prolonged storage.

You may use kinased oligos (or is it "oligoes"?) or you may kinase the annealed
duplex prior to ligation.

Hope this helps.

Hiranya.


Quoting EK <[Only registered users see links. ]>:



--
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---

Thanks Hiranya,
For annealing with the vector, I will not use ligase and rely solely on
annealing of complementary sticky ends on the vector and the inserts. The
reason for it is that I believe it will reduce the anount of self ligated
vector. I do get clones of self ligated vector if I use ligase, despite the
noncompatible ends.
Emir

""Dr. Hiranya S. Roychowdhury"" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ].edu...
with
each.
source
hot
degrees.
C
annealed
annealed
will
plasmid?

Why don't you dephosphorylate your vector? Noncompatible ends do not mean that
you will ot get "self-ligation". What you see is inter-molecular concatenation
with non-compatible ends (not really self-ligation. which generally refers to
an intra-molecular event). If you take the phosphates out, then there will be
no ligation between vector molecules.

I am not sure about ligation without ligase. Never done it!

Hiranya

Quoting EK <[Only registered users see links. ]>:



--
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---

In article <NzXPb.24$[Only registered users see links. ]>, "EK" <[Only registered users see links. ]> wrote:

Here is what I have done quite a few times:

Mix the two oligos at 10 microM each in 20-100 microL in TE. Stick
the eppendorf in the 300-500 ml beaker, microwave the whole thing
until boils and leave to cool at rt until only no longer warm to
touch.

Make serial dilutions of the above in water: 1:10, 100, 1000, 10000.
Mix 1-2 ul of each with 1 ul of linearized vector (concentration
not important within the "usual" range), ligate 15 min rt,
transform. One of the conditions always gives 10-100X over
background (no insert).

I've never tried not using ligase but if all you want is to get the
construct made, using ligase will do no harm.

DK

>Thanks Hiranya,

What I've generally done for cloning oligos like this is to NOT
kinase the oligos, then use a few levels of annealed oligo insert in
the ligation reaction, like 3X, 10X, and 30X molar excess over
vector. Since the oligos aren't phosphorylated, you don't recover
clones with multiple inserts, but you can still favor getting clones
with insert by having a lot of it in the ligation reaction.

As for self ligated vector despite non-compatible ends, it has been
argued in this newsgroup before that this can be due to incomplete
digestion with both enzymes, with religation of single cut vector a
favorable event. If there is another enzyme that cuts between the
two you are using for your cloning, you could actually cut vector
with three enzymes to reduce this possibility.

What vector are you using for this cloning? Is there color
selection. This would allow you to ignore religation events due to
single cuts.

Mike
--

Michael L. Sullivan, Ph.D
Research Plant Molecular
Geneticist
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5397 Phone
(608) 264-5147 Fax
---

""Michael L. Sullivan"" <mlsulliv@facstaff.wisc.edu> wrote in message
news05200f12bc374c28626f@[192.168.80.1]...
the
It is an unusual cloning strategy. The plasmid vector is pre-cut with the
blunt-cutting restrictase and then the sticky ends are generated by T4
polymerase in the presence of only one nucleotide. The polymerase removes
one strand all the way until it gets to the first letter matching the
nucleotide in the reaction. In my case, 15nt sticky ends are produced on
both ends of the linearized vector. Inserts encode short peptides, and once
inserted into the vector, the peptides can be expressed in fusion with an
enzyme. It is basically an ELISA-based screening system for peptide ligands.
No color selection.

Anyway, I think you guys answered my initial question and I appreciate your
advises. I just should try several dilutions and ratios of insert and vector
to find optimal conditions for one or two samples and use those throughout.
It would not be feasible though to do dilutions for every case, because the
idea is to clone and screen hundreds of peptide sequences at a time at a
time, possibly using lab automatics.

Cheers,
Emir

"EK" <[Only registered users see links. ]> wrote in message news:<sPnQb.50$[Only registered users see links. ]>...

Hello!

Emir, why dont you dephosphorylate your vector like Hiranya suggested,
and just ligate the oligos into you vector? Anyway ligase wont
interfere with any of your downstream processes i think, fully agree
with DK.

I have some suggestions about T4-removing single strand DNA, make sure
you are doing it at low temperature, i dont remember how low it should
be, some 14 or 12 degrees, check it out in a tech.. cause this enzyme
is such a reactive pacman that could eat everything you need to remain
in you vector:-))))

my recipe for doing oligos is ideal for gel shift assays, and i dont
see any reason why it shouldt work with cloning...

Have a nice cloning,

Drew