4 replies to this topic

[JessiBelli89]

Hey everybody,

i just graduated as a bachelor of science in biology. Now i'm working in the labratory as a labratory assistent and got the task to prepare in situ probes. But I have already trouble with the first steps

I ligated a 1kb fragment into pCRII vector and transformed One Shot TOP10 cells with this ligation mix. But unfurtunately I just got many blue colonies and a few white colonies which doesn't have my 1kb insert. I tried it several times but I got always the same result.

Can somebody help me with my problem? I would be very pleased because I don't have a clue

Sorry for the bad english..and thanks for lots of answers

Jess

[jerryshelly1]

Can you provide more information.  You are using a plasmid with an X-gal gene so you can immediately eliminate all white colonies from your selection procedure.  Did you verify the presence of your insert within your plasmid before transformation.  Do a PCR with primers specific for both insert and vector.  You can also confirm by digesting your vector with a couple of RE to obtain unique sizes.

[JessiBelli89]

Hey, at first thanks for ur answer. The plasmid I'm using carries a lacZalpha fragment with a MCS. Therefore integration of my insert causes a frameshift and all my positive colonies are supposed to be white. Afterwards I isolated the plasmids and digested the construct to get a band for my Insert and the vector.

I tried to check for the insert befor transformation by digestion with the same enzyme (EcoRI) I am using for analyzing my clones.
Unfortunately the concentration was to low to visualize clear bands on the gels. I just saw very slight bands for the plasmids but definitely nothing for the insert.

But returning to ur answer. I will definitely try the version with the primer if available tomorrow. Otherwise I repeate the digestion protocol with a higher amount of my ligation mix.

I will also proceed some control reactions explained in the kit. I will see what's coming out of this.

I think too that the problem is the ligation. What would u suggest do optimize my ligation. I proceeded ligation with a 300bp fragment before and ligation and transformation worked fine. Do u think increasing the concentration of the PCR-Prod could do the job?

Thanks for the reply, kind regards Jess

[jerryshelly1]

I honestly don't know.  Double check your plasmid and Insert concentrations.  Maybe adjust your vector to insert ratio or try a different ligation condition.  Is your insert blunt or sticky ended?

[JessiBelli89]

I am using a vector with single 3' deoxythymidine (T) residues and the PCR-Product is amplificated with DreamTaq Polymerase which suppose to add single deoxyadenosine (A) to the ends of the PCR-Products. Therefore the PCR Product is allowed to ligate with the vector.