Using single restriction enzyme SalI for cloning to a binary vector. How I will - Please its really very urgent...help me...help me.. (Sep/29/2007 )

Hello all,
I would like to clone a gene to pGREEN binary vector in SalI restriction site. The gene is already in a vector and I can digest out it from SalI site. I really did not able to find any other suitable restriction sites. My qustions are..

1, If I clone on single restriction site how I will know it got ligated in the proper direction??? (start to stop direction)
2, In general how should be the direction of cloning???(Please refer the attached image of T-DNA) should it from KpnI to SacI (start to stop) or SacI to KpnI???????

Please someone kindly guide me......

1. If you conducted a colony PCR to screen for your desired clone, only colonies carrying plasmids with the orientation (as defined by the primers) will amplify. Thus orientation is a natural product from the screen. Conformation (or an alternative screening method) of the clone is conducted by restriction digest. In this situation, you need to make 2 cuts. The first cut is within the vector at a known location and the second cut, cuts your insert asymatrically. Thus depending on orientation, the two cuts sites can be either closer or further from each other. This in turn means that depending on orrientation, the fragment cut out is either larger or smaller.

2. Orientation would depend on your intend; what do you want to do? Is there a promoter in the vector? Do you want to express your gene? What do you want to do with your gene? Will futher ligation work be done? If so, is one orientation more helpful then another?

Finally one a personal note; SalI is a bad enzyme. It has a bad reputation. DNA cut by SalI do not ligate well. (It can be done, just the failure rate feels higher then normal.) Are you certain that SalI is your only option? Is it possible to PCR amplify your vector, adding more convinient restriction sites on the 5'end of the amplification primers

Thank you very much perneseblue, You always help me a lot. I know the Sal I site is bit problematic but unfortunately I dont have any other option.
Regarding orientation, this is a final step cloning to T-DNA region of pGREEN to over express in plants. This gene is a cassette with its own promoter and even one GFP gene is integrated inside this gene. I am little bit confused with the orientation or direction which given on the T-DNA sketch (lac Z orientation). So if you can please explain the orientation stuff little bit, I think it is really important for proper expression of any gene...Thank you...

The orientation is important in relations to restriction site and promoters.

Simply put it. if your gene is place backwards to a promoter, you don't get a gene product. And the gene's relationship to local restriction sites are important if you want to futher manipulate your plasmid by adding more fragments. If the restriction sites are ordered wrongly further cloning can be awkward.

From the picture provided, orientation appears unimportant. All that is present is a multiple cloning site in an empty binary vector with no important features. More so that your gene has its own promoter/terminator sequences. It can function on its own.

Will there be further manipulation conducted? Aside from the binary reaction? Adding more sequences?

If orientation does not matter, personally I will pick the orientation when both genes (your gene and the LacZ gene) are in the same orientation. I looks better.

Thank you very much perneseblue. If I understood correctly if my transgenes, promoter-gene-terminator orientation is right, it dosent matter how it cloned to binary vector.??? This is really very basic I know but I am not much experienced in this field..Thank you very much..

The orientation is important in relations to restriction site and promoters.

Simply put it. if your gene is place backwards to a promoter, you don't get a gene product. And the gene's relationship to local restriction sites are important if you want to futher manipulate your plasmid by adding more fragments. If the restriction sites are ordered wrongly further cloning can be awkward.

From the picture provided, orientation appears unimportant. All that is present is a multiple cloning site in an empty binary vector with no important features. More so that your gene has its own promoter/terminator sequences. It can function on its own.

Will there be further manipulation conducted? Aside from the binary reaction? Adding more sequences?

If orientation does not matter, personally I will pick the orientation when both genes (your gene and the LacZ gene) are in the same orientation. I looks better.