gateway system problem

[biotech]

hallo all,

 

I have a problem with understanding the gatewaysystem.

 

 

I have a vector that I want to turn into a destination (expression vector).

 

Now I have made blunt ends and the reading frame of my vector ends with a triplet of bps.

 

So I have:

 

vector sequence .... TTA and here I want to introduce the gatewaysystem TGT rest of vector sequence.

 

Now, if I follow the invitrogen information it states I should use the reading frame A (for N tag), but I do not understand how the reading frame stays ok if the resulting attb sites are 25 bps long!

 

The genes I want to introduces into the gateway system (between the attb sites) are ORFs , so they start with an ATG codon right away.

 

So this is how I see it after LR reaction:

 

Expression vector sequence ...... TTA 1/2 EcorV (3bp) attB1 my ORF attB1 1/2 EcorV (3bps) rest of my expression vector sequence....

 

Now the attB1 site is 25 bps , so I dont see how the reading frame stays as it should stay!

 

I mean: TTA ATC (=1/2 EcorV) 25 bps ATG .... attB1 1/2 EcorV ....

 

those 25 bps => they will alter the reading frame and my ATG will not be in frame anymore!

=> it will be 21 bp attB1 site GGC TAT G.. ...... (T from tha attB1 site and the AT from the ATG start codon...

 

So I am not sure this will work out...

 

Or ?

 

 

[chadn737]

It depends on what you used for an entry vector. In some cases you should add 1-2bps to your primers to put the clone in frame. What are you using for an entry vector?

Edited February 15, 2014 by chadn737