Hi Shadariah,

DH5alpha is a strain for high yield plasmid DNA production, not proteins.

It has distinct features for that purpose. As an example: endA genotype to ensure low endonuclease content for high plasmid yield. RecA for reducing unwanted recombination of the plasmid.

BL21DE3 is intended for protein production it lacks an endoproteinase

for improving the protein yield and quality. Your plasmid is most likely under the control of an inducible promoter,depending on the system you are using you have to induce protein production by IPTG or use an autoinduction medium.

I don't want to go into details but the choice of the right strain is absolutely critical . Take a bit time to get familiar with the protocol provided by your provider and the underlying principles and you will spare yourself from a lot of frustration. A basic understanding of the bacterial genotypes is necessary. You can find them among others here:  http://openwetware.org/wiki/E._coli_genotypes. There is an explanation of the genotypes at the end of the list. 

I hope this helps a bit. 

Regards 

David 

Hi Shadariah,

DH5alpha is a strain for high yield plasmid DNA production, not proteins.

It has distinct features for that purpose. As an example: endA genotype to ensure low endonuclease content for high plasmid yield. RecA for reducing unwanted recombination of the plasmid.

BL21DE3 is intended for protein production it lacks an endoproteinase

for improving the protein yield and quality. Your plasmid is most likely under the control of an inducible promoter,depending on the system you are using you have to induce protein production by IPTG or use an autoinduction medium.

I don't want to go into details but the choice of the right strain is absolutely critical . Take a bit time to get familiar with the protocol provided by your provider and the underlying principles and you will spare yourself from a lot of frustration. A basic understanding of the bacterial genotypes is necessary. You can find them among others here:  http://openwetware.org/wiki/E._coli_genotypes. There is an explanation of the genotypes at the end of the list. 

I hope this helps a bit. 

Regards 

David 

Hi there,

If you use pET plasmid serie then protein expression will only be efficient in a strain expressing T7 RNA polymerase. The simplest to do so is to host your plasmid into a DE3 strain (containing the gene encoding for T7 RNA polymerase under the control of lac promoter). So efficient expression will occur after IPTG addition (produced RNA polymerase being entirely dedicated to T7 promoter on the pET vector) or if the only carbon source is lactose.

Dominique Liger's explanation is appropriate.  pET vector have T7 promoter and that promoter is not recognised by e.coli RNA polymerase so DE3 gene( which codes T7 RNA polymerase) is incorporated in  the genome of BL21 Strain and the resultant strain is reffered as BL21 (DE3) strain.

Thank you to all. =) its really help me now.

yesterday i do the transformation into the BL21DE3 using the same kit  and today it seem that nothing grow on the plate. im using the following method ;

1.mix the the cloning reaction to 10ul with ( 2ul 5x in fusion HD enzyme premix,1ul linearized vector, 5 ul purified pcr fragment, 2 ul deionized water) 

2. incubate the rxn 15 min,50 degree celcius, then place on ice

3.continue transformation.....since im using BL21DE3, so i followed this method

      a) 5ul of cloning rxn + 200 ul competent cell ,gently flish

      b) incubate on ice 20 minutes

     c) heat shock: 42 degree celcius, 45 sec n do not shake

  d) return the tubes to ice for 2 minutes immediately

      e) add 800ul LB BROTH (we dont have soc medium) to the tubes (contain cells transformed with ligation rxn)

      f) incubate 1.5 hours ,37 degree,shaking 150 rpm

      g)spin down at room temperature 1000g, 6 minutes

     h) removed out 800ul of supernatant

      i) resuspend the remaining (~200ul)

     j) about 100ul were used to spread plate (2 plate)

     k)incubate 37 degree,overnight

this is the method i used yesterday.. resulted no grow

Dear Shadariah

To answer to your question and helping you, I need some more information abot your work:

1) would you please and say some more about the pET vector? which pET series have been used? How you prepare it for ligation?

2) How you prepare your insert (PCR product)? Do you digest it with any restriction enzyme(s) or not? If yes, which enzyme?

3) Which and how much antibiotic did you add to LB medium?

4) I am not familiar with the kit which used for ligation . Would you please and send some technical information ( like catalogue No. and company name) about this kit.