Using a vector.....

liberal

Im intergrating an operon into a vector plasmid that will hopefully transform recipent bacteria....

What role do primers play in this? My superviser keeps going on about primers, all I know is that you use primers for PCR, I don't want to ask him because I feel it might make me look stupid

Thank you in advance!

Tree

Well, you'll want to perform pcr on the segment you want to put into the vector, so you can have buckets of the stuff for transforming your bacteria (unless you're making a library). So your segment to insert will need appropriate primer sites to amplify it, and appropriate sites for restriction enzymes to cut it for insertion to your vector.

TBH you're better off asking him now, rather than in a few weeks when you're completely stumped. Blame the summer break/midterm break/distracting stain on teh floor for your lack of remember why primers are so important to you.

Kevster

Indeed, just ask him now. I don't see what the deal is with seeming as if you re 'stupid', because you quite simply WON'T seem stupid. You WILL seem stupid if you mess it up later on and have wastd lots of money and time. These molecular chemicals and molecule are VERY expensive. You don't want to be using them ad lib.

r3nu4l

You're inserting the whole operon?

Okay, well you need to know the genetic sequence of the operon from start to end.

Then you need to design 'primers' for it so you can do the PCR as Tree said.

Primers are basically a shortish string of nucleotides (referred to as oligonucleotides).

They need to be designed specifically for your sequence of operon DNA. They are basically reverse complements of the DNA you want to copy.

You need two primers to copy your operon. One will be designed to copy the operon from the start of the DNA, the other designed to copy from the end of the DNA.

Have a look at this and this.

Now, talk to your supervisor! S/he is there to help you. If s/he's too busy, find a post-doc and ask them for a crash-course in PCR and vector usage.

If you are inserting this operon into a vector there's a couple of things you need to bear in mind.

Some vectors only accommodate certain sized PCR products, make sure the one you choose suits your product size (use an Invitrogen or Promega or other company catalogue to get this info).

You may have to design your primers to not only be reverse complementary to the DNA you want to copy but also to have a restriction site on each end so that it integrates exactly into the vector in the right reading frame.

The vector you choose, also needs to be suitable for the viral or bacterial host you will be replicating it in.

So, read the company catalogues, talk to your supervisor and also talk to others around you. There's no such thing as a stupid question in science. A good scientist asks questions all the time.