no colony

[plasmamembrane]

Hello,

 

I am trying to clone a 1.1Kb gene into 5.7Kb plasmid. It seems straight forward but I got no colony. Below is my protocol.

 

Insert preparation:

The insert was generated by PCR. RE sites ( BamH1 and HindIII ) were designed on 5' end of primer. I also generated extra 4 bases for efficient cutting.

The PCR was fine. I got a sharp band on gel. I cut the gel and purified it. The product was doubly digested ( BamH1-HF and HindIII-HF

from NEB in Cutsmart buffer for 15 min ). Then I gel purify it again.

 

Vector preparation:

Vector was also double-digested with two REs. I gel purify the DNA after digestion.

 

Ligation:

 

80ng of vector was used for ligation. Vector/insert ratio was 1:3.

ligation enzyme: NEB T4 DNA ligase

time: RT for 1hr then 65degC for 10min for inactivation

 

transformation:

 

DH5alpha ( competency: around 10^8 cfu/ug DNA )

2uL of ligation mixture was used for transformation

heat shock: 1min at 42 degC

recovery: 45min in SOC

 

incubation O/N at 37 degC on LB-antibiotic plate, but no growth.

 

Need your help.

 

Thanks a lot

[Rajnish Kaushik]

put some sugar in it just try it

[CharonY]

put some sugar in it just try it

 

Sorry, but this is precisely the kind of answer that annoys me. On the one hand it is nonsensical (what type of sugar, at which step and why the heck at all?) but is stated almost as a fact. While it is rather easy to dismiss in this case, in others it may very well confuse readers.

 

That being said, quickly looking over the protocol and there does not appear to be huge issues. Two things that I noted are 1) A run of four bases is a bit on the short side (six or more tends to be safer).

2) Gel purification sometimes can result in low and damaged product (the latter if it is being illuminated on UV for too long), but other than that it is pretty standard. In my hands I tend to have more consistent results with column based purification.

 

There are a suite of troubleshooting experiments that one could do to ensure that things work out as expected on every step (e.g. transfer intact vector, digested vector, re-ligated vector, etc.). It may be the case that either your digest/gel purification went very well or the yield was low as I would have expected at least a tiny amount of re-ligated vector.

[sona_p]

I have a question before I give you any suggestion.

1) Are these two sites in your vector present in MCS of the plasmid?

 

If yes, then don't gel purify your vector, it will avoid loss of plasmid during the process.

 

2) If you are just deleting few bases from the PCR product, I would recommend skipping the gel purification step.

3) Ligate it for more than 12 hrs/over night at 4 degrees

4) Have a negative control where there is no insert added. In that way, you would get some idea about the background ligation.

 

Reply if you have any more questions

 

 

[chadn737]

That's a lot of unnecessary gel purifications. I never do gel purification unless I need to separate two large fragments, such as an insert from another vector. Furthermore, T4 ligase generally works best at ~16C spread out over several hours. I also don't see the point of inactivating the ligase, I have never done this. Just transform it without this step. Consider doing a longer digestion as well. 15 mins is pretty short, depending on how much you have in your reaction.

 

There are really a lot of possibilities. Sometimes cloning just doesn't work and you hit your head against the wall and have to start from scratch.

I also used to run some of my ligation reaction on a gel alongside the digested vector. If the ligation reaction had a larger product I knew the ligation worked and I always had a successful clone.

[CharonY]

Well, for many applications a certain amount of purification is necessary e.g. to get rid of dimers though a simple column clean up is often more than efficient. Especially if you do lots of cloning (i.e. plates) you would like to have a certain amount of purity (again, I am no fan of gel purifcations myself). Inactivation of ligases is sometimes done for electroporation. Ligase binds to DNA (which you can see as a band shift in gel) which reduces electroporation efficiency. But I agree, for heat shock transformation it is pretty much a non-issue.

[Dekan]

put some sugar in it just try it

 

As annoyed CharonY points out, that suggestion sounds nonsensical. On the other hand, doesn't recent research in another biological field seem to show that stem-cells can be produced, simply by putting skin-cells or blood-cells into a bath of weak citric acid for half an hour. Suppose last year someone had suggested trying that - "put some acid in ". What would've been the response - "Nonsense - how could that possibly work" ?

[CharonY]

That is not analogous as in this case the system is very clearly defined (i.e. enzyme, DNA, buffer system). In the case of the mice blood cell you have a) a more complex system and b) the idea behind it was to induce stress response in the cells. One of the responses was pluripotency.

Originally, it was known from plant cells that this may occur, so it was not a matter of throwing random stuff at cells.