Dear Xuezhi,

If the vectors you recover do not have your insert, the most likely culprit is the BglII digestion. The BglII enzyme is not very robust.

We use NEB enzymes. According to the manual BglII has 75% activity in buffer 2, but I find this to be much lower. On the other hand SpeI has a reported activity of 25% in buffer 3, but I find that it actually cuts just fine in this buffer. Therefore I perform all BglII/SpeI double digestions in buffer 3.

Another way to be sure you get a good double digestion is by using pDM vectors with a GateWay cassette. For a C-terminal GFP with G418 resistance that would be pDM353. Upon digestion with BglII/SpeI the 1.7 kb GateWay cassette drops out of the vector and that gives a nice band shift on gel. I have found this bandshift to be the most useful property of GateWay vectors.

Furthermore I would recommend switching to hygromycin as a selectable marker. Selection with hygromcyin faster and transfected cells are robustly resistant against hygromycin, whereas cells under G418 selective pressure always appear somewhat sick and have an increased doubling time. The hygromycin resistant C-terminal GFP fusion vector with GateWay is pDM450 and this is by far the most used vector in our lab.

Kind regards,

Douwe Veltman


-----Original Message-----
From: DICTY on behalf of Xuezhi Zhang
Sent: Fri 24/06/2011 17:37
To: [log in to unmask]
Subject: [DICTY - 564] pDM323 cloning
 
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]