Dear Zhi Qi,
I agree with Peter. You must check whether the ssDNA and dsDNA are
phosphorylated. We also had extremely low ligation product, but when we
re-phosphorylated the fragments to be ligated using polynucleotide
kinase, we got quite good ligation products.
Rijuta
>> Have you checked that the ssDNA and dsDNA segments have phosphates at
> the appropriate ends? If you don't have the phosphates in the right
> places, you won't get any ligation.
>> Peter
>>>> Zhi Qi wrote:
>>> Dear all,
>>>> I want to ligate a ssDNA with a ~2-kbp dsDNA with a 15-nt overhang. The
>> ssDNA oligo has a 60-bp hairpin construct with a 15-nt overhang at 5'
>> end. These two 15-nt overhang on ssDNA and long dsDNA are designed to
>> complimentary to each other.
>>>> I added ssDNA and dsDNA together with enough T4 DNA ligase (NEB), and
>> ssDNA:dsDNA = 100:1 (I just hope ligation efficiency is high). I
>> incubated the sample at 16 C for 12 hour and then 65 C for 20 min to
>> inactive T4 ligase.
>>>> From the 1% agarose gel, I can get ~2.1-kbp band which means the ssDNA
>> + dsDNA. However, when I used gel extraction kit (QIAGEN) to cut the
>> band out and purify it again, and run another gel to check it, this
>> ~2.1-kbp band lost. I guess ~2.1-kbp band I got just an annealing
>> product (15-nt overhang is long enough to anneal each other, right?),
>> but the ligation did not work. It is because my dsDNA is too long
>> (~2-kbp) to block the T4 ligase to bind on the ligase position?
>>>> Another thing I know is that the yield of single strand ligation is
>> lower than the normal ligation (two strand ligation)
>>>> I will be grateful if you can give me some idea and suggestion.
>>>> Waiting for your reply.
>>>> Best Regards,
>> Zhi Qi
>> Biophysics Department
>> UIUC
>>>>>>> ------------------------------
>> Message: 6
> Date: Wed, 03 Sep 2008 10:21:53 +0000
> From: Christian Praetorius <prae from gmx.net>
> Subject: Re: BAC Protocol
> To: methods from net.bio.net> Message-ID: <6i76q3Fpe2aeU1 from mid.individual.net>
> Content-Type: text/plain; charset=us-ascii
>> "sanjiban chakrabarty" <sanjiban.c from gmail.com> wrote:
>>>> I am working with BAC DNA and I have faced lot of problem with BAC minipreps
>> as the yield is always very low and the BAC DNA is contaminated with E Coli
>> genomic DNA.
>>>> We have either been using a modified protocol for the Qiagen Midi prep
> Kit (http://www1.qiagen.com/literature/render.aspx?id=450&tp=9) or the
> PhasePrep BAC DNA Kit from Sigma (#NA0100). Yields were usually quite
> low, but since we prepared the BACs only to transfer them between
> different bacterial strains, this was ok for us.
>> Christian
>>
