I am interested in hearing what others have to say about this as we have struggled with the problem for some time. The problem seems to be when two enzymes in the vector are close together, but if you look at the sites and the enzyme overlap requirements, you are led to believe it should work, but it does not. We find usually you recover the original vector, implying both sites did not get cut, even though you can show that both enzymes work fine individually under the same conditions. Choosing sites farther apart usually fixes the problem, but you don't always have that option. I have wondered whether the first enzyme there actually stays associated with the cut site and sterically blocks the other enzyme for getting at the DNA. I haven't found any literature which says this is or is not possible. If it were happening, then doing separate digests with a phenol step in between should fix the problem, but we have not tried that yet. Dave
On Jun 24, 2011, at 2:42 PM, Petra Fey wrote:
> Hi Xuezhi,
>
> I think it's a high chance that either your vector or the PCR products are not cut properly. Did you do double digests? In the vector, the two restriction sites are very close and migh inhibit eachother, plus SpeI and BglII also prefer different buffers, so it might help to digest sequentially. Also, for you PCR products, do you have 3-4 nt overhang beyond the restriction sites to cut efficiently?
>
> Just some thoughts. Good luck!
>
> Petra
>
>
> ------------------------------------------------------
> Petra Fey
> Northwestern University
> Biomedical Informatics Center/NUCATS
> 750 N. Lake Shore Drive, 11-175-C
> Chicago, IL 60611
> USA
> [log in to unmask]
> ------------------------------------------------------
>
>
>
> On Jun 24, 2011, at 11:37 AM, Xuezhi Zhang wrote:
>
>> Dear all:
>> Before I make the next try of cloning, I think it's better to ask you first for some advices.
>> I am trying to clone 3 different genes into pDM323 vector respectively, and the shortest is around 1800bp, the longest is about 3600bp. The PCR works fine and the BglII & SpeI double enzyme cut also works well both on the insert and the vector. But I already tried several times of ligation and electroporation, and there were very little colonies on the plates (at most 20 colonies for the 1800bp gene cloning), and those colonies are just negative ones after checking by PCR, miniprep and enzyme cut. I already increased the vector/insert molar ratio until 1:10, and tried different ligation conditions, such as 16 ℃ overnight, room temperature 15 min and 30 min, but never improved.
>> This cloning stuff already trapped me for almost one year. I really appreciate someone of you could give me some suggestions to clone these genes into pDM323 vector.
>> Thank you very much! And have a nice weekend.
>>
>> --
>> Xuezhi Zhang
>> Department of Biochemistry
>> University of Geneva
>> 30 quai Ernest Ansermet, Sciences II
>> CH-1211-Genève-4, Switzerland
>> [log in to unmask]
>
Dr. David Knecht
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)
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