news.100.johanneswinkler at spamgourmet.com
Tue Jan 3 09:03:42 EST 2006
Using your approach, I never had the need to phosphorylate oligos for
linker insertion (of course the vector was not dephosphorylated). There
is no need even to anneal them, just add a sufficient molar excess
(>500) of the linkers ... By avoiding
For a reference, see "One-Step Insertion of Oligonucleotide Linkers or
Adapters to DNA Using Unphosphorylated Oligonucleotides", Chulho Kang
and Masayori Inouye, BioTechniques Vol. 15, No. 4: pp 659-668 (Oct 1993).
It worked great for me every time using plain T4 ligase (no "quick"
ligation kits or additives). Good luck.
> In article <mailman.607.1135724894.29584.methods at net.bio.net>, Mark Blenner <markblenner at yahoo.com> wrote:
>>Please post the following:
>> Hello Everyone,
>> Looking for some advice with my ligations. I have purchased two oligos that
>>just need to annealed - they have my two stickey ends - AflII and BseRI (this
>>is downstream of the recognition sequence - sequencing has told us how to
>>design the sticky end).The insert is 48 bp. They have been annealed and
>>verifyed on a gel. The vector is about 3.5kb cut with AflII & BseRI and gel
>>purified. Concentrations determined using UV-260- Ligation performed with the
>>NEB Quick Ligation kit using ratios of insert to vector of 1:1, 3:1, 5:1,
> Not much use of going by these formal numbers. You'd be better off
> coivering a wider ranges of ratios. I always anneal at 25 uM oligos then
> ligate 1 ul of it at 1:10, 1:100, 1:1000, 1:10000 ratio. Depending on
> vector/insert combinations (and scores of factors I cannot readily account
> for), I've had best results at 1:100 through 1:10000 dilutions.
>>Transformed into purchased GC5 compotent cells and plated according to
>>the resistance of the plasmid. I never get colonies.
> A rule of thumb: if things are working properly, you ALWAYS have to have at
> least *some* colonies - even if you've done everything wrong :-)) E.g., there
> always has to be a background!
> What does "no insert" control give? No colonies? Then these are the likeliest
> 1) Your competent cells are crap. Do a control with another ligation/pure
> vector to make sure efficiency is sufficient.
> 2) your vector is damaged (e.g. UV fried, nuclease/star activity somehwere,
> vector was cut with a wrong enzyme by mistake). Run the vector on a gel and if
> it looks good, redo the prep, doing single cut at a time, gel purification
> and omitting any phosphatase step you might have.
>>Control plasmid and cell
>>controls verify that the plates kill non transformed cells and the control
>>plasmid transformed bacteria survive. I have tried this with PCR products and
>> different plasmid and restriction sites and no colonies either.
> Are you saying that you can't get any ligation of any kind to work?
> Then the problem is much more global and you should investigate
> very seriously. Notmally, ligations are a no brainer.
>>frustrating - another person in the lab gets her ligations to go
>>relatively easy, from the same batch of buffer and ligase.
> If you can get this person to help you, he/she can help to roubleshoot
> your problem by picking up where you left things - thus eventually
> zeroing on the problem step.
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