i always subclone and sequence before putting the insert into its final
vector (e.g., pDM323).
if the vector is not being cut properly to allow for insert ligation, i
would expect much more than 20 colonies. both bglII and speI have short
cutting times and from what i can see in pDM323 the sites are separated by
4-5 bps...this minimal overhang can make it difficult for an enzyme to
recognize the site if it has been cut. under these conditions, sequential
digests seem to work better in my hands. cut with one...clean it on a
column, then digest with the next enzyme. i would use bglII first because
it cannot be heat inactivated. the in a double digest one of the enzymes
might be mis-cutting the site...hence no ligation of vector to itself or
with insert and few colonies.
second, if you're trying to pcr the fragment after it has already been
subcloned, you can pretty much rule out toxicity of the fragment. but i
would typically use 6 NTs upstream of the primer restriction site to
ensure RE cleavage...3 can work, but not as efficiently. third...if you
are going to go the PCR route...order primers with BamHI sites on each
primer...cut pDM323 with BglII and purify...purify insert, ligate, take an
aliquot of the ligation, cut it with BglII...this will linearize
re-ligated vector but not insert ligated vector....transform cells and
screen colonies by directional pcr. XL10 gold cells are best if worried
about vector size but your vector+insert size doesn't seem too big.
mike
Xuezhi Zhang wrote:
> Actually, I did the subcloning at first, and the 3 genes are subcloned
> into
> pJET1.2 vector and sequenced correctly, but just the later steps not work
> when I want to clone the gene from pJET1.2 into pDM323. So I tried to
> clone
> directly from the PCR products which are still not working well.
>
> 2011/6/24 Luna, Elizabeth <[log in to unmask]>
>
>> We always go the TA and subcloning route. It’s much more reliable
>> than
>> restriction digests of PCR products. Another advantage is that you can
>> sequence-verify your insert before the final cloning step and know that
>> none
>> of your destination constructs has a PCR-induced point mutation. With
>> genes
>> ≥1800 bp, the odds are that 1 of every few PCR products will have a
>> mistake
>> somewhere, at least based on our experiences; even high-fidelity
>> polymerases
>> introduce errors with enough PCR cycles.
>> Beth Luna
>>
>>
>>
>> On 6/24/11 4:20 PM, "David Ratner" <[log in to unmask]> wrote:
>>
>> I'm not familiar with going higher than, say, 3:1 to force in an insert,
>> irrespective of vector size. Recently a student new to the lab had
>> trouble
>> cloning two PCR products, even though the vector was clearly linearized
>> and
>> phosphatase treated. We had put plenty of nucleotides 5' to the
>> restriction
>> sites on the primers, so assumed that shouldn't have been a limitation.
>> One
>> effective alternative, although it takes an extra step, is to TA clone
>> the
>> PCR product into some appropriate blue-white T vector, than pop it out
>> again
>> via primer-designed cutting sites in order to introduce it into the KO
>> vector. Although slower on paper, this has been working very well for us
>> of
>> late.
>> David Ratner
>>
>> Xuezhi Zhang wrote:
>>
>> OK, thank you all. it seems that my problem is due to the digestion of
>> my
>> vector, hopefully. So I am gonna try a sequential digestion. And another
>> question, for this kind of cloning, the vector itself is already quite
>> big
>> (nearly 7600bp), and my genes are at least 1800bp, so would it help if I
>> use
>> high insert/vector ratio, for example 10:1 (0.3 pmol insert, 0.03 pmol
>> vector)?
>> Thank you.
>>
>>
>> On Fri, Jun 24, 2011 at 9:43 PM, Petra Fey <[log in to unmask]>
>> wrote:
>>
>>
>> When I did a lot of cloning in the past, yes, I did sometimes sequential
>> with heat inactivation and a phenol/chloroform step. Also, after cutting
>> my
>> PCR products, I usually gel-purified, or at least precipitated using 4M
>> Ammonium acetate to remove the primers; any columns to purify them might
>> or
>> shoud work too of course. It's just that all these things matter in they
>> add
>> up.
>>
>> petra
>>
>>
>>
>> On Jun 24, 2011, at 2:07 PM, David Knecht wrote:
>>
>> > 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 <tel:860-486-2200>
>> > 860-486-4331 <tel:860-486-4331> (fax)
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>
>
> --
> Xuezhi Zhang
> Department of Biochemistry
> University of Geneva
> 30 quai Ernest Ansermet, Sciences II
> CH-1211-Genève-4, Switzerland
> [log in to unmask]
>
----------------------------------------
Michael A. Myre, PhD
Center for Human Genetic Research
Massachusetts General Hospital
Harvard Medical School
Richard B. Simches Research Center
185 Cambridge St., CPZN 5.612A
Boston, MA 02114
Ph. 617-643-5536
Fax 617-726-5735
[log in to unmask]
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