1) Your plasmid could be self ligating. Try dephosphorylating plasmid and then use for cloning.

2) Your insert may be too big. You can try using stbl2 cells or grow cells at 30 degrees.

3) Try colony PCR before incubating in LB for miniprep.

4) Always have positive and negative controls as recommended by others.

Can you get the plasmid isolation to work with empty vector (no insert)? If so, then your insert may be toxic to the DH5a E. coli. If the empty vector fails too, then it would seem that something is wrong with your reagents (such as LB contaminated with detergent, or at the wrong pH).

I have the same problem with my colonies ( I have a lot of white colonies) but after Miniprep, no plasmid found!!! (I'm using pGEM T vector)

Did you perform a negative and positive control (according the positive control see Daniels answer)? It might be, that your e.coli somehow get resistant, or your antibiotic got bad. So the possibility arises that you pick false positive colonies which did not take up your plasmid: In combination with Daniels suggestion you should figure out the problem.

Good luck

As Danniel and Guido suggested, control experiment is really important, especially when ecountering any problem. I set up control every time while transformation, because there are so many possible factors which would effect the final result.

BR

1) Your plasmid could be self ligating. Try dephosphorylating plasmid and then use for cloning.

2) Your insert may be too big. You can try using stbl2 cells or grow cells at 30 degrees.

3) Try colony PCR before incubating in LB for miniprep.

4) Always have positive and negative controls as recommended by others.

All the above suggestions are reasonable. Do your control transformations to rule out bad reagents. if it still does not work, change the host cell. After several passages these strains start behaving strange. Use newly aquired cells. As Mritunjay suggested you can also change to Stbl2 or other host strains recommended for larger construct transformation. Do recovery at 30 degree celcius and incubate your plate at 30 degree celcius. You can also go down on antibiotics concentrations a bit.

Good luck

Anirudh

The Antibiotic i used is good one. The thing i didn't understand is how a cell survives in Antibiotic without plasmid?

By the way thank you all for your kind suggestions.

In addition to the preview suggestions, for difficult transformation you can use transformation tubes instead of eppendorf-tubes. Sometimes it helps a lot. (If i were you, first i would change the coli strain.)

Asif- bacteria growth in the presence of antibiotic indicates resistance. In almost all cases, this is due to a resistance gene that inactivates your antibiotic, and such genes are typically encoded in a plasmid. In rarer cases, these resistance genes can be incorporated into the host genome or the bacteria can pick up a mutation that renders them resistant to the antibiotic. In addition, not all bacteria are susceptible to the same antibiotics, which is why I am concerned that you may have contamination by another microbe that is not E. coli. Any of these latter scenarios would lead to bacteria growth without a plasmid.

Hi

I have the same pb

your plasmid may be to large to use simple heat shock transformation... You may have to use electro-competent bacteria.

Hi,

First, as Daniel said, if you can get your empty vector easily but have problems after the cloning, you may have an issue with the toxicity of the insert. For the LB and the plates used for the transformation, there shouldn't be a problem since the hygromycin-containing media is provided with the plasmid when you buy it. Also the size of your vector is definitely not a problem. I transform in routine vectors of 15 or 16kb in chemically-competent DH5a and it works perfectly fine. Indeed, this vector contains an origin of replication completely suitable for any E.coli strain so even the most basic DH5a should be fine (subcloning DH5a from Invitrogen for example).

Your problem of transformation efficiency could be linked also to a problem of cloning and/or kit used for the cloning. For instance, I would do a control of the ligation to be sure that you have a difference between the insertion and the self-ligation of the plasmid. I don't know what kind of cloning you do but with some kit like "Quick ligation kit" from NEB, there's PEG in the reaction and it inhibits all the transformation so you have to clean your DNA before the transformation. Also the protocol of transformation is important for the chemically-competent cells. Indeed, the incubation on ice of the DNA with the cells is a key moment (usually 30 minutes but every 10 minutes you shorten this step results in 2-fol loss of efficiency) as well as the recovery at 37C (1h is good but every 15 minutes you shorten results in 2-fold loss of efficiency; SOC is much better than LB to resuspend; shaking is better for recovery than nothing) after the heat shock.

Finally, because it's a cloning, tough apparently, I would definitely clean/concentrate the reaction, using the Zymo Research kit for example to transform as much as possible.

Good luck.

You need to mix gently your DH5 alpha cells when adding the vector using pipette tip!!

can you look into more precisely what would be the best restriction enzymes to be used to release GFP first ? then clean your ligation reaction result ... then see what happen