Suggestions on Research design re: Cloning the promoter region and measure induc - Molecular genetics, protocol (Jul/02/2006 )

I know there are lots of experts here, i would like to ask for your suggestion regarding the research design and protocols to be utilized to solve the following objectives. I appreciate every help that i could have. Thank you in advance

OBJECTIVES

a)clone the promoter of a certain gene
b)measure the level of expression and confirm induction
c)create a knock out strain

-arvinsign-

first step - find the promoter. that's the tricky part. or will you just clone a bunch of DNA upstream of the start site and use the knockout clones to study changes in expression? what, specifically, is your overall goal? the question you have asked is very very broad. do you already have the upstream region on a plasmid, or will you start with chromosomal DNA and the gene's sequence and go from there? there are many, many ways to do this. what have you done so far to research methods?

-aimikins-

QUOTE (aimikins @ Jul 3 2006, 05:13 AM)

Hi! Thanks for replying. ill be working on a gene that is yet to be characterized in terms of functions. Its a bacterial gene that proves to be important in regulating virulence in a close family strain of the bacteria that il be using. My professor wants me to study this, thats y im asking for suggestions. I guess my promoter is an alternative promoter bec induction happens during period of stress or stationary phase. If ill start with the gene sequence, what do i need to do?Thank you in advance.

-arvinsign-

First step, buy Sambrook and Russell; Molecular Cloning - Third Edition. Cost about £139. If you can't then locate a copy of Maniatis (lab bible - though now way out of date and superseded by the above book).

Second step, read it!

-Doc_Martin-

arvinsign - I am with Doc Martin. there are so many steps involved and it's very important to understand the 'why' at each point and be certain you have it right before you move on. I would definitely thumb through that entire book, even if you are getting guidance from your prof

and, ask your prof questions; I bet you're not expected to hand him a paper in two months with no help...as long as you show initiative as well they usually like that sort of thing...perhaps find a mentor in your lab who is skilled at these techniques you will need

-aimikins-

Thanks aimikins and Doc martin your advice is well appreciated. Im familiar with the book, and i happen to borrow that from our library for a couple of times now, but im thinking of doubling the effort and time reading it. i guess i really need somebody in the lab who can teach me these techniques. Thanks

-arvinsign-

Definitively, we MOST read, the sambrook, other books and a lot of papers.
I don't think it's your case, but.....

specially, when we are new in this field, we need someone near who can help and guide! Some time we don’t even know where to look for…..
It’s awful when you're very new and your only help is the sambrook and no one also!!!
Specially when there are a lot of ways to do something, and you (ALONE) have to choose… and you don’t know which could be the best for you.
I remember, when I was new and was alone, and I read… digest the appropriate amount of……. Or digest with the adequate enzyme…... Or elute in the proper volume……. And I wandered what??? Which one?? How much???? Now it sound very simple and some times, ridicule , but in that time.. it was awful!!!!!

-aztecan princess-

QUOTE (aztecan princess @ Jul 12 2006, 04:53 AM)

I remember, when I was new and was alone, and I read… digest the appropriate amount of……. Or digest with the adequate enzyme…... Or elute in the proper volume……. And I wandered what??? Which one?? How much????

Now it sound very simple and some times, ridicule

, but in that time.. it was awful!!!!!

very well said princess, thats exactly the point. the case is im new (in some of the techniques), nobody cared (almost) to help me in the lab to teach me protocols. I know that reading my books, journals, searching other resource materials is my reponsibility and ive been doing that. But reading is not enough, students need guidance. I believe that all expert researchers nowadays have come to this point in their lives too. I saw this bioforum and i felt quite relieved because i want to believe that there are many good souls in here who wants to share their expertise.i have faith in this forum and im learning and at the same time enjoying a lot.

-arvinsign-