Can N-terminal or C-terminal tags be attached to a Gateway® Entry clone?
To have an N-terminal tag, the gene of interest must be in the correct reading frame when using non-TOPO® adapted Gateway® entry vectors. All TOPO® adapted Gateway® Entry vectors will automatically put the insert into the correct reading frame, and to add the N-terminal tag you simply recombine with a destination vector that has N-terminal tag.
To attach a C-terminal tag to your gene of interest, the insert must lack its stop codon, and be in the correct reading frame for compatibility with our C-terminal tagged destination vectors. Again, TOPO® adapted Gateway® Entry vectors will automatically put the insert into the correct reading frame. If you do not want the C-terminal tag to be expressed, simply include a stop codon at the end of the insert that is in frame with the initial ATG.
Generally, you need to choose a destination vector before you design and clone your insert into the Entry vector. This will determine whether you need to include an initiating ATG or stop codon with your insert.
Answer Id: 3950
Can you go directly from a pENTR™/D-TOPO® reaction into an LR Clonase® Reaction without first purifying the DNA?
In most cases there will not be enough pENTR™ vector DNA present to go directly from TOPO® cloning into an LR reaction. You need between 100-300 ng of pENTR™ vector for an efficient LR reaction, and miniprep of a colony from the TOPO® transformation is necessary to obtain that much DNA. However, if you want to try it, here are some recommendations for attempting to go straight into LR reactions from the TOPO® reaction using pENTR™/D, or SD TOPO®, or pCR®8/GW/TOPO® vectors:
1. Heat inactivate the topoisomerase after the TOPO® cloning reaction by incubating the reaction at 85C for 15 minutes.
2. Use the entire reaction (6 uL) in the LR clonase reaction. No purification steps are necessary.
3. Divide the completed LR reaction into 4 tubes and carry out transformations with each tube. You cannot transform entire 20 uL reaction in one transformation, and we have not tried ethanol precipitation and then a single transformation.
When attempting this protocol, we observed very low efficiencies (~10 colonies/plate). So just be aware that while technically possible, going directly into an LR reaction from a TOPO® reaction is very inefficient and will result in a very low colony number, if any at all.
Answer Id: 3953