Hi,

The criteria very much depends on a few things some of which are:

1. what system you are expressing in (yeast, E. coli, Mammalian, insect etc)

2. What do you want to do with it: Do you intend to purify or just express (perhaps transient/stable etc)

3. do you need a tag for purification/identification: Hist, myc, FLAG etc. although these can be included in PCR products etc

4. Strength of the promoter: i.e. T7 in pET system for E. coli is a strong promoter but may not be applicable for your protein. CMV for mammalian system is also strong for Hek cells. Do you need tight control of expression.

5. other additions: in mammalian system you can look at vectors with further additions: IntronA sequences from CMV, episomal expression, directed stable cell line expression (see Flp-In from invitrogen).

6. The nature of your protein will to a certain extent determine what system you use and thus vector: big mammalian proteins with lots of glycosylation and disulphide bonds may not express too well in E. coli.

In our experience in E.coli and mammalian it is best, if possible, to try and few combinations of vectors with slightly different characteristics. We use both stable cell line development as well as transient in mammalian systems (see pCEP4 from invitrogen). We have used different vector combinations in E.coli with good success.

good luck

It mostly depends the purpose of your cloning. If one is interested in protein expression then the cloning vectors are different from those used just for any DNA fragment. The size of the DNA fragment also matters in the choice of the vector and also whether the purpose is just cloning a DNA fragment or even sequencing. So in short the purpose of cloning decides the right vector

Also the number of copies. Different plasmids could has different number of copies.

It is also important the epitope (bigger, smaller, Ct, Nt, for purification, for IP, for fluorescence, inmunohistochemistry, etc)

Its also important to think of down-stream applications. Do you just want to clone something from PCR, than TA-Vectors are useful. If you want to overexpress proteins, you need the right promoters (mamalian vs. eukariotic) and probably tags.

Thank you very much to all

Hi,

The criteria very much depends on a few things some of which are:

1. what system you are expressing in (yeast, E. coli, Mammalian, insect etc)

2. What do you want to do with it: Do you intend to purify or just express (perhaps transient/stable etc)

3. do you need a tag for purification/identification: Hist, myc, FLAG etc. although these can be included in PCR products etc

4. Strength of the promoter: i.e. T7 in pET system for E. coli is a strong promoter but may not be applicable for your protein. CMV for mammalian system is also strong for Hek cells. Do you need tight control of expression.

5. other additions: in mammalian system you can look at vectors with further additions: IntronA sequences from CMV, episomal expression, directed stable cell line expression (see Flp-In from invitrogen).

6. The nature of your protein will to a certain extent determine what system you use and thus vector: big mammalian proteins with lots of glycosylation and disulphide bonds may not express too well in E. coli.

In our experience in E.coli and mammalian it is best, if possible, to try and few combinations of vectors with slightly different characteristics. We use both stable cell line development as well as transient in mammalian systems (see pCEP4 from invitrogen). We have used different vector combinations in E.coli with good success.

good luck

Smaller size, selectable markers and Multiple cloning sites are most important.

MCS/ ori replicaion in vector is major concern if you need only DNA copies say for cloning and sequencing. If you are interested to expression of the gene of interest then MCS/replication site/ RNA polymerase promoter site are of major concern.

thank you very much to all specially Ian

if you want to have basic ideas about cloning you can refer to book GENE CLONING of TA brown

Since you have mentioned cloning vector, I assume that its not for expression. Theoretically, vector characteristics involve 3 fundamental requirements, small size, presence of unique restriction sites, and markers for recombinant selection. You will have to see which vector possesses the restriction sites for the enzyme you propose to use. In case you are cloning PCR products you can always build the restriction site of your choice into the primer and clone in the available vector. Normally, blue white screening works absolutely fine. For cloning and sequencing, I have found the pKS vectors to be very good.