basic questions regarding expression vector work - (Jun/09/2008 )

Hi guys ,

Its me again bothering u all with questions.
I have to work with expression vector now . I have to express zebrafish protein . I am planing to purchase pcdna 3.1 for this work. I want to know a few things.
Is this a good vector? Is it easy to get high expression using this vector.
why there are two kinds, pcDNA 3.1 (+) and (-) ?? How to decide which one I am supposed to use.

another big question , my ORF has kozak in it but it does not has a G after ATG which I read is important for expression. If I include an extra G in my primer wont it change the whole protein sequence ? How am I supposed to that.
I am new to this work, with nobody around to teach me properly. is there any guideline for protein expression work. i dont want to do silly mistakes and end up cloning again and again.

Thanks guys
will appretiate this help.

regards
VAni

Well I can answer the first part,

pcDNA3.1 is a good vector for high expression in mammalian cells, should be easy to get high expression of your protein, assuming the size isn't too big etc.
The two kinds 3.1 + and - refer to the multiple cloning site (MSC) restriction enzyme order. basically + goes one way and - is the opposite direction. So you need to choose the vector which allows you to clone in your sequence in the right orientation for expression. For example, if the + one has the enzymes in the order of EcoR1, Sal1, Not 1, Pst1 and your gene is flanked by EcoR1 and Not 1 (5' to 3') then use the pcDNA3.1 (+), but if its Not1 and EcoR1 (5' to 3') then you need pcDNA3.1 (-).

can't really help you with the Kozak thingy, however I'm fairly certain you don't want to put an extra G into the sequence as then you'll get nonsense and no protein expression- the ORF (open reading frame) will change with the addition of a single nucleotide.

Hope that explain things a little

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