cDNA library and molecular biology in general- am I in too deep - (May/08/2008 )

Hi,
As my user name suggests I'm new to this field. My background is in general biology and ecology (of fish), but as part of an overseas post doc (lasting 1.5 to 2 years) I have been thrust into molecular biology. What I thought was originally suppose to be a small section of my research in collaboration with a collegue who specialises in molecular biology, has resulted in 9 months of molecular biology research and increasing pressure to become an expert/ focus my research to this field (including helping write a review). The research is basically targeted at isolating a particular gene and then examining its expression level using real time PCR.

To date, after 9 months we have made little progress. A fragment of the gene was isolated using primers designed from the target gene which was originally isolated from another fish species. However, after many attempts the 3' and 5' ends could not be extended. Therefore, a cDNA library was constructed (using probes designed from the above mentioned primers) and sequenced but despite using various approaches the sequence result were no good so I have been informed that I should repeat the library again from scratch.

Over the past 9 months my technique has gradually improved, and with help from websites such as this I have started to understand the background a little more. However, my colleague and supervisor think that this should have been ample time to get a grasp of molecular biology (i.e. identify what goes wrong, why, what the next step is etc) and are basically starting to blame the lack of results on me and keep on telling me to study more (basically by reading books- no courses etc or help has been offered). Despite the fact that I have stated that I don't want this to be the main focus of my research (molecular biology wasn't even in the research proposal approved by my home country and the one I'm in now). Admittedly, some experiments had to be repeated probably due to my poor technique but these have not caused any great delays.

Anyway, now that I have given some background into my issue, I have the following questions.

1) How long should it roughly take to isolate a specific gene (maybe a bit like asking the length of a piece of string, but a range is ok)? i.e., would 1 year be overly long in this circumstance?
2) Given the target gene has been isolated in another species (from a completely different family) is the design of primers from this gene the only way to obtain primers or probes? Should I consider another method of designing primers or screening? Shouldn't I consider other screening methods (and/ or probes) before starting the library again from scratch?
3) In addition I was wondering as we could not extend the 3' and 5' ends of the fragment isolated using these primers, is it then suitable to use these primers to design the probe?
4) Is it realistic to expect the suggested level of understanding of molecular biology theory and techniques in this time frame (ie. 6-9 months)?

I apologise if these questions are a little basic for this discussion but any advice will be greatly appreciated.

I woud also like to make comment on the excellence of this forum, there are so many people out there that are willing to offer advice and assistance. Seems like a great bunch of people- maybe I should consider changing fields!!!

Sorry to hear about your troubles! Do you have anyone in your lab to help you with your molecular biology?

What did you use to extend the 3' and 5' ends?

If your original primers for your gene are ok, it shouldn't take too long to get the full length sequence. Did you want the full length before you moved onto looking at expression levels?

When I had to work out the full coding sequence for a gene I used a BD RACE (rapid amplification of cDNA ends) kit to extend my original primers - I then cloned the longest sequence into a vector and then sequenced it to get the 5' information. A few weeks work if everything goes well - not that it always does in molecular biology!

Sorry I can't write a longer reply - I just woke up

Hi Clare,
Thanks heaps for your reply. Unfortunately, there is only the one colleague that I previously mentioned. I'm not in a molecular biology lab- the supervisor also has little or no experience in this field (hence why I value this website).

We used Takara 3' and 5' full race core sets. In addition we tried using both gotaq and phusion hot start high fidelity DNA polymerase. We plated on pGEM- T Easy vectors. I think we tried a few other options as well. The fragment we isolated was about 400bp and we were trying to get the full length before examining expression level. But from your msg I get the impression that the expression levels can be examined without obtaining the full length (maybe in some circumstances)- is that correct?