Hi,

I believe that more details are needed ;)

Did you prepare your plasmid by cloning a PCR product?

No: if you only transformed bacteria with prepared plasmid, try to isolate the construct (mini scale) and digest it with insert-flanking restriction enzymes to check the insert size.

Yes: did you get a proper 2.3 kbp product in your PCR before cloning?

   Yes: if you had a proper product and you digested it with restriction enzymes during construct preparation, then perhaps you have an internal cleavage site? or enzyme star activity?

   No: optimize PCR reaction. For example if you amplify the region that contains GC-rich regions add betaine to your reaction. Try other polymerases.

Best,

Lukasz

Hi,

I believe that more details are needed ;)

Did you prepare your plasmid by cloning a PCR product?

No: if you only transformed bacteria with prepared plasmid, try to isolate the construct (mini scale) and digest it with insert-flanking restriction enzymes to check the insert size.

Yes: did you get a proper 2.3 kbp product in your PCR before cloning?

   Yes: if you had a proper product and you digested it with restriction enzymes during construct preparation, then perhaps you have an internal cleavage site? or enzyme star activity?

   No: optimize PCR reaction. For example if you amplify the region that contains GC-rich regions add betaine to your reaction. Try other polymerases.

Best,

Lukasz

I agree with Lukasz.. I suggest the more reliable way to understand it correctly is to do restriction digestion ananlysis. Use an enzyme in multiple cloning site and one in between the insert check you get the expected size. Do this with two to three restrcition digestions to have a correct idea. Importantly check whether your primers  are specific . Hope this helps good luck !!

There could be due to:

And this is true if the former advices are done, and you really have shorter insertion in vivo.

Hello,

I agree with Danila, we had seen in our lab, problem of getting a clone with a gene and its endogenous promoter.

Mostly, there were no clones with right sized insert and if there was one or two with correct size, then it was because of some or the other mutation in the gene, leading to the loss of toxicity. This despite the fact that the endogenous promoter was not thought to be expressed in E.coli. There are some promoters that can express in E.coli, so you do need strong terminator and strongly repressed promoter.

Try to get the sequence of 1500 bp insert, you will know, what part is cloned and is not toxic, so either you are losing the promoter and partial gene to get 1500 bp, or may be some recombination even is occurring, leading to some internal deletion.

Hope it helps. Best,

Simran

All the suggestions above are valid. I really advise you to sequence the 1500 bp insert to analyze that fragment. Maybe you are dealing with an unexpected recombination event.

^^^ what he said. Sometimes there's a limit to how much you can guess, second guess and triple guess. Sometimes you just have to "Just DO it". Sequence the fragment, then tell us what you found.

Good Luck

G

I agree with all suggestings, I just want to ask you how did you get the plasmid? I guess that you inserted your gene between two enzyme restriction site. did you sequence it before transformation? did you use these same restriction enzymes to insure that your gene is 1.7 kb?

Hope it help 

Also, I would back up first: did you have your insert in the plasmid already or did you PCR your fragment from the organism of interest. I agree with above comments, sequence your fragment first of all. 

Using REC - genotype E coli strain allows  you to avoid recombination or expression of the gene of interest with genes of E coli during transformation. You did PCR using plasmid primers. I inform you that polymerase may create mutation (single base pair mutation) (possibly deletion) which can leads to truncated sequence of DNA. It's better to sequence the product of PCR to see what's wrong. Good luck 

Thank you everyone for your advise. I did PCR previously and I got a 1.7kb gene product. Then I digested it with Apa1 and Hind111 which are compatible with my plasmid. I cloned it into my digested pBBR1MCS-2 plasmid with promoter and did transformation into S17-1. When I did PCR screening, the size only 1.5kb. I tried to extract the plasmid and digest again using same REs, nothing is being digested. So I suspect my gene does not go into the plasmid, what should I do?

If you get the proper product in your PCR, than its already almost halfway to succeed ;)

Now it is the matter of digestion:

It seems that you insert only a c.a. 0.7kb from your PCR product into a plasmid (0.7+0.8promoter=1.5 PCR product with plasmid primers).

Some hints:

- are you 100% sure you have no internal restriction sites for ApaI or HindIII in your 1.7kb PCR product?

- HindIII has some star activity and may cut not perfectly matching sequences when overincubated. Run DNA electrophoresis on your PCR product following digestion to check if you still have your 1.7kb product.

- ApaI is short-living at 37oC and is suggested to be used in 25oC. Did you perform sequential digestion?

For ApaI/HindIII info follow that link:

https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder?enzyme1={E462690D-A5A6-4A21-B7B6-3E81BBE93580}&enzyme2={873FF478-6285-44A2-A62F-8DF1659E4EF3}

Could you please tell how you inserted your promoter into your pBBR1MCS-2 plasmid? And which plasmid primers do you use for transformants screening?

Best,

Lukasz

Did u checked the cutter and non cutter sites of your plasmid before designing your primer for gene?

In my opinion, this is a contamination accident.  I suggest you, try to redo the cloning experiment  from digestion of  the vector and fragment, re-ligate, and retransform. after each step, confirm the fragments size by gel.  BTW, where is your gene from? RT-PCR?

Did you try sequencing the plasmids you recovered? This might tell if you managed to clone something resembling the desired insert. If you do see something that has a resemblance to what you are expecting, perhaps the sequence got rearranged by the E.coli DNA repair/recombination systems during plasmid propagation. This is very common if the sequence contains repeats, and/or has biased base composition. The only solution is to try different E. coli strains, e.g. recA-, recBJ-, etc. and/or use a cloning plasmid vector with a lower copy number

Now that Philip Shaw mentioned it, I tried different E.Coli strains and I ended up using "One Shot® Mach1™-T1R Chemically Competent E. coli". I am sorry for the publicity but it actually saved me a lot of trouble.

You can try to do PCR to check if you have your insert if you are having difficulties with  digestion. 

When you do check digest and run on gel, make sure you run a few controls, may be your empty vector digested/undigested. as well. 

I would try an extension time of two minutes. If that doesn't work, look online and find the best melting temp. based on the nucleotide ratios. Also a longer ligation time should also help.

Best

In my lab's experience, we have found some AT-rich sequences can only be cloned when the plasmid is transformed into the CopyCutter cell from Epicentre. On the other hand, sequences with inverted repeats are stable in SURE2 cells, but not recA- strains. I do not receive anything from these companies to promote their products; this is just my experience as their customer  

Hi, 

If I have not followed all information about your question and if you still trying to resolve this problem, is there any additional restriction sites in your insert (1.7 Kb PCR product, check by screening the sequence) for the two restriction enzymes (Apa1 and Hind3) you used to digest your plasmid? if yes, then you should try with different REs. if no, you can sequence your plasmid (successful clones with insert) after extraction using one of the primers of your gene of interest not of the plasmid. I hope it will help.