10 replies to this topic

[cusycon]

I'm trying to ligate my PCR product about 300 bp with vector about 10 kb.
The ligation was done at 1:3.
The antibiotic selection is erythromycin about 50 ug/ml.
I got this plasmid from a particular paper and it said concentration of erythromycin is 350ug/ml in E.coli and 5ug/ml in Lactobacillus sp.
But when I try to just transform the empty plasmid(with no insert), there is no colony was found.

So I,then find the optimal concentration of erythromycin by streaking the transformation(empty vector) and competent cells on the same concentration of the plate.
And It was found that about 50ug/ml is the best. (with transformated colony grows and competent cells don't grow)

After that I try to transform the ligated plasmid with ratio 1:3 and then transform.
The colonies grow so so many!!! and the size is very very small.
I cannot isolate even one colony. and The result of transformation is the same when i try with other genes.

I really don't know why it is happen like this.
and I really don't know that something wrong with concentration of antibiotics or not.



Please help me......I'm stuck with it for 6 months

[pesji]

Could you give the reference of the paper ? I never seen any plasmid using Erythromycin resistance genes  

[HomeBrew]

Erythromycin resistance is inducible -- you might try plating the transformants first on plates containing a low concentration of the antibiotic, and then streaking the growth for single colonies to plates containing a higher concentration of erythromycin.

[phage434]

You can always restreak a transformation plate for isolated colonies.  Or you can simply plate lower amounts or dilutions of your transformation.  But I'm concerned that you have not gotten your insert.  When you say 1:3 ratio, do you mean a molar ratio or a weight ratio?  It should be a molar ratio, which in this case would be a very small amount of your insert.  What are your enzymes?  How are you cutting? purifying? ligating? transforming?

[cusycon]

phage434, on Nov 5 2009, 08:33 PM, said:

You can always restreak a transformation plate for isolated colonies.  Or you can simply plate lower amounts or dilutions of your transformation.  But I'm concerned that you have not gotten your insert.  When you say 1:3 ratio, do you mean a molar ratio or a weight ratio?  It should be a molar ratio, which in this case would be a very small amount of your insert.  What are your enzymes?  How are you cutting? purifying? ligating? transforming?

Thanks for your all suggestions


My enzymes are BamHI and EcoRI. I incubate 37c about 2 hours. I also run the gel for check whether my vector is completely cut or not. And the result from band is one clear band for plasmid digestion.

Both of insert and vector are purified by gel purification and then gel electrophoresis is performed. The banda are clear.

Ligation i used 1:3 with molar ratio. Ligation was done in 10 ul of total reaction by NEB T4 ligation overnight 16C.

Transformation used conventional method. CaCl2 heat shock 1 min.

The plate is prepared by mix Em at temp lower 45C

---------------------------------------

Yesterday, I test my hypothesis of antibiotics.

i divided into 3x2x4 groups depend on concentration of Em x reaction that used x amount of plate after transformation


1. 50ug/ml
plate competent cell alone plate100  -  bacteria can grow - smeary +++++
plate plasmid (without cut) plate50 -   bacteria can grow - smeary +++
plate plasmid (without cut) plate100 -   bacteria can grow - smeary ++++
plate ligation reaction plate50        -   bacteria can grow - smeary +++
plate ligation reaction plate100       -   bacteria can grow - smeary +++

2.100ug/ml
plate competent cell alone plate100 -  bacteria can grow - smeary ++++
plate plasmid (without cut) plate50 -   bacteria can grow - smeary ++
plate plasmid (without cut) plate50 -   bacteria can grow - smeary +++
plate ligation reaction plate50        -   bacteria can grow - smeary ++
plate ligation reaction plate100       -   bacteria can grow - smeary ++

3.200ug/ml
plate competent cell alone plate100 -  bacteria cannot grow
plate plasmid (without cut)plate50 -   bacteria cannot grow but in some position(5%) also has thin smeary
plate plasmid (without cut)plate100 - bacteria cannot grow but in some position(5%) also has thin smeary
plate ligation reaction  plate50       -   bacteria cannot grow but in some positions(5%) also has thin smeary
plate ligation reaction  plate100      -   bacteria cannot grow but in some positions also(5%) has thin smeary


I also try to plate the ligation after transformation only 10ul in LB 90 ul (this in order to dilute) - so smeary

So after I look at my result, i think it is ok for the first 2 experiments but for 200ug/ml I don't think it is normal.

1. the thin smear may be because the volume of plate is too much.
2. my plate technique is not good.

But If my test is correct that means the optimal antibiotic concentration is about 100-200ug/ml.


from my reference paper Em concentration is 350ug/ml. and the other papers (3 papers) with the same plasmid use 300 ug/ml. So I think the competent cell effiency (commercial or home made) is also relevant.


So, now I don't know which point should focus on??

[HomeBrew]

Streak some of the growth from the ligation reaction plate at 50 ug/ml to plates containing 200 ug/ml.

As I said before, expression of erythromycin resistance is not always constitutive -- sometimes expression has to be induced by exposure to low (non-selective) concentrations of erythromycin.  Once induced, cells carrying the inducible gene are resistant to high concentrations of erythromycin.  Erythromycin is bactericidal -- if the resistance gene is not constitutively expressed and is inducible, plating cells directly to high concentrations of erythromycin will kill them.

Two other questions: Did you allow a period of non-selective growth before plating the transformants?  What vector are you using?

[phage434]

What is the plasmid you are using?  It sounds as if either the cells you are using are already Erm resistant, or the plasmid is not working.

Your gel purification worries me  -- be careful  about UV damage.

Your transformation protocol is also a concern.  CaCl2 is great for subcloning, but not for initial transformation of ligation products.  Consider using highly competent cells.

[cusycon]

HomeBrew, on Nov 6 2009, 07:37 PM, said:

Streak some of the growth from the ligation reaction plate at 50 ug/ml to plates containing 200 ug/ml.

As I said before, expression of erythromycin resistance is not always constitutive -- sometimes expression has to be induced by exposure to low (non-selective) concentrations of erythromycin.  Once induced, cells carrying the inducible gene are resistant to high concentrations of erythromycin.  Erythromycin is bactericidal -- if the resistance gene is not constitutively expressed and is inducible, plating cells directly to high concentrations of erythromycin will kill them.

Two other questions: Did you allow a period of non-selective growth before plating the transformants?  What vector are you using?

I got your point.

I used pIAlac. I don't know that whether you have heard about it or not.
Thing is I 've no map of this plasmid because the author doesn't give it to me. I don't know why but I already request him 3 times for 5 months ago

[cusycon]

phage434, on Nov 6 2009, 08:53 PM, said:

What is the plasmid you are using?  It sounds as if either the cells you are using are already Erm resistant, or the plasmid is not working.

Your gel purification worries me  -- be careful  about UV damage.

Your transformation protocol is also a concern.  CaCl2 is great for subcloning, but not for initial transformation of ligation products.  Consider using highly competent cells.

Thank your for your suggestion.
I'll try other technique may be electroporation.

[phage434]

pIAlac is Erm resistant to 300 ug/ml according to the  paper Perez-Arellano02, Structural features of the lac promoter affecting gus expression in Lactobacillus casei.  The E. coli origin of the vector is a p15A origin, which is low copy number, and may mean you are having difficulty preparing plasmid DNA.  There is more than enough information in that paper to allow full sequencing, which would be the first thing I would do if I were serious about working with the plasmid.

[christy]

phage434, on Nov 9 2009, 12:07 AM, said:

pIAlac is Erm resistant to 300 ug/ml according to the  paper Perez-Arellano02, Structural features of the lac promoter affecting gus expression in Lactobacillus casei.  The E. coli origin of the vector is a p15A origin, which is low copy number, and may mean you are having difficulty preparing plasmid DNA.  There is more than enough information in that paper to allow full sequencing, which would be the first thing I would do if I were serious about working with the plasmid.

Try 1:10 ratio of vector:insert. I suceeded ligation of 1.3 kb insert in 10 kb vector