Changing the vector - (Aug/05/2014 )

Feel free to ask any other questions you have down the line. :)

Hello again,

questions here,

1-does all the primers need to have 5' phosphate group? 

2- At which stage I need to sequence, after amplify the gene, after cloning the gene into the new vector or later after making the point mutation?

Sorry if questions sound very naive but I don't have even chart for the cloning experiment yet. All I need is the final mutated cell line to start my spectroscopy analysis!

In general if you are going to digest the PCR product, then you don't need to 5' phosphorylate the primers. If you were doing something like Qickchange then I would say it might be better if you did have the phosphates.

I would sequence once you have it inserted into the new vector. You will also need to sequence after the site directed mutagenesis, so that you can confirm the presence of the point mutation.

Hi again which competent cell kit to order for heat shock protocol ?

There are many choices. Our standard competent cells are NEB10B cells, a version of DH10B, but many others would be suitable. You can also make good ones yourself here:

http://openwetware.org/wiki/TOP10_chemically_competent_cells

what about Top10. I found it already in our freezer ?

It's essentially the same as DH10B or NEB10B.

Do you have access to an electroporator? If so, I would suggest making electro competent cells. They are easier to make (for me anyway), require no extra chemicals other than 10% glycerol and tend to give better efficiencies. The down side is you have to either dilute or purify your ligation before transforming so it doesn't blow up on you (arc) but that's all I use.

Or yeah, you can use ones you already have :-P

I have more questions:

what after ligation  GOI to the open vector can I sequence directly or i should transfer it to e-cloi amplify the plasmid before sequencing?