After Gibson assembly or T4-Ligation obtained colonies do not grow anymore - (Dec/23/2015 )

Hi,

I used NEBuilder® HiFi DNA Assembly Cloning Kit (Gibson assembly) for the cloning of 3 fragments (vector, 7100bp,  and two inserts, 283 and 115bp) with 30bp overlaps.

This seemed to work fine: I transformed the provided cells from NEB, I obtained clones.

FIRST ANOMALY: the colonies were somehow strange, not so contrastfull, diffuse...simply strange.

I picked some of them, replated them in a secondary LB-Agar-plate with ampicillin and performed in parallel a colony PCR. I even let sequence the PCR-Product. Result: band-length in the Agarose gel and sequence are correct. The sequence contains both inserts and parts of the vector on both sites.

SECOND ANOMALY: the bacterial clones don’t grow anymore in any conditions.

Neither on Agar nor in liquid LB cultures. Neither with nor without Ampicillin. Neither in LB nor in SOC medium. Neither on my plates nor on the plates of a neighbour-group.

The control transformations with the control plasmid grow normally.

I used the commercially available competent cells from NEB and from Clontech with the same result.

From the assembly reaction I amplified by PCR the recombination product of the two inserts: 421 bp. I repeated the assembly with only 2 fragments (vector and single insert). After transformation: some strange colonies, no growth.

I digested the combined single insert with the same enzymes as the vector was linearized. I purified the digestion product and performed T4-Ligation. After transformation: some strange colonies, no growth.

I tried to reduce the amount of transformed DNA. Same results.

Do you have an explanation to this strange phenomenon? What shall I try more? Choose another vector? It’s a quite frustrating situation.

The vector is for lentiviral expression, contains eGFP as reporter gene and the aim is to clone a promoter upstream to GFP.

Thank You for the help.

Luca on Wed Dec 23 10:36:55 2015 said:

Hi,

 

I used NEBuilder® HiFi DNA Assembly Cloning Kit (Gibson assembly) for the cloning of 3 fragments (vector, 7100bp,  and two inserts, 283 and 115bp) with 30bp overlaps.

 

This seemed to work fine: I transformed the provided cells from NEB, I obtained clones.

 

FIRST ANOMALY: the colonies were somehow strange, not so contrastfull, diffuse...simply strange.

 

I picked some of them, replated them in a secondary LB-Agar-plate with ampicillin and performed in parallel a colony PCR. I even let sequence the PCR-Product. Result: band-length in the Agarose gel and sequence are correct. The sequence contains both inserts and parts of the vector on both sites.

 

SECOND ANOMALY: the bacterial clones don’t grow anymore in any conditions.

Neither on Agar nor in liquid LB cultures. Neither with nor without Ampicillin. Neither in LB nor in SOC medium. Neither on my plates nor on the plates of a neighbour-group.

 

The control transformations with the control plasmid grow normally.

 

I used the commercially available competent cells from NEB and from Clontech with the same result.

 

From the assembly reaction I amplified by PCR the recombination product of the two inserts: 421 bp. I repeated the assembly with only 2 fragments (vector and single insert). After transformation: some strange colonies, no growth.

 

I digested the combined single insert with the same enzymes as the vector was linearized. I purified the digestion product and performed T4-Ligation. After transformation: some strange colonies, no growth.

 

I tried to reduce the amount of transformed DNA. Same results.

 

Do you have an explanation to this strange phenomenon? What shall I try more? Choose another vector? It’s a quite frustrating situation.

 

The vector is for lentiviral expression, contains eGFP as reporter gene and the aim is to clone a promoter upstream to GFP.

 

Thank You for the help.

I do not really get it.

You confirmed that everything went well by doing the PCR and the sequencing?

So you must have some plasmids ?

So can you not use those plasmids to retransform rather than using old colonies?

You explanation is a bit confusing.