perneseblue

First you have to answer a question

Do you plan to work  with cDNA (without introns) or a full size  gene?

Human  gene tend to have splice site variant and contain regulatory elements within their introns. A cDNA will not be able to capture these facets.
On the other hand full size eukaryote tend to be very big and not easy to manipulate. cDNA tend to be much smaller.

Depending on your answer, you may use one these as a template

a plasmid from a cDNA libraries
a plasmid from a BAC libraries
raw human DNA.

So where can you get a template?

You may either purchase the template DNA or obtain it from a lab working in the field.  Using pubmed find out who has publish work on your protein of interest  and email them. Invitrogen and Open Biosystems are examples of places where you may purchase a BAC libraries. A google search will turn up more places which sells cDNA or BAC libraries.

Working with raw human DNA is not easy. Stick with libraries.

could we have the sequence of your primers?

The annealing temperature is rather  high.

Also what is the expected PCR product size?

well it depends if the plateau phase has been achieve after that many cycles.
If both PCR reaction mixes have reached the plateau phase than mixing the two could not change the concentration. Since the concentration of PCR product would be identical.

However if the plateau phase has not been achieved, then the reaction mix with the fewer number of cycles would logically has less product, and thus a lower concentration. Pooling the two would increase the amount of PCR product present but decrease the concentration.

I would add 5 cycle even 10 cycles is rather few. I use 25 cycle to amplify my PCR product.

Aside from not being as efficient as possible, I don't see a problem pooling the two.

CTAB is a detergent.  It is as dangerous as SDS.

Thus the solution is safe so long as you don't drink it or wash your eyes in it. Same as any other detergent.

And similar  to any powdered detergent, work with CTAB powder should be done in a fumehood. Breathing in detergent powder of any kind is harmful to your lungs.

As far as harmful chemicals go, I would call CTAB a bench top chemical, especially the solution. CTAB powder can be kept in a closed container on the bench, but I would only open the container in a fumehood.

The protocol I use for conventional alkaline lysis (Sambrook) on mini-preps, adds 100ug/ml of RNAse A  to Solution I.

HomeBrew, on 07 August 2010 - 06:43 AM, said:

To summarize, we disagree on two point.

1-The degree that humanity plays in the this global warming trend.

2-How humanity should allocate our limited and finite resources.

Why are those two points of contention important? I have come to realize it is because they overlay a more basic question, "Do we have sufficient time to adapt our civilization or do we need to buy time."

If we have sufficient time to adapt our civilization, I agree, we should spend our finite resources adapting. If humanity does not have enough time, it would be logical to expend some resources buying time, so we can complete the adaption process.

I stand on the side of "We don't have enough time". People take a lot of time to get their act together.  Humanity has to get through the recognition stage, the denial stage (I think we are here), the anger stage, the acceptance stage, the long planing stage and finally the implementation stage.

Knowing people's propensity to procrastinate and dash madly to meet dead lines, the same would probably happen during this challenge. And worryingly, we will only know when the deadline has arrived when our crops fail, our seaports flood and shortages of goods due to disruptions from single point failures in the global supply chain. And yes, I recognize not all system failures will hit at the same time. But I believe those short few years will not provide enough time to adapt. Nor would the warning signs be recognized as such.


I also stand on the side that "Humanity do make a significant impact to the global warming trend."

Thus humanity can "buy" time to adapt by moving to green technology which has reduced emission of carbon dioxide. However I was thinking a step more than that. I was thinking of actual expenditure of resource/energy to remove carbon dioxide from the atmosphere (my preference is to carbonize wood and storage of the charcoal produced underground). And yes, the dreadful solar shield idea with sulfur dioxide in the upper atmosphere would come under the idea of "buying time". Dreadful because knowing human behaviour, once the solar shield is up, people will stop bothering and make no attempt to adapt civilization. And when the shield goes down, we will get hit with all the changes in an instant, without even the few decades to adapt that we would receive with this warming trend.

TTT, on Jul 4 2010, 04:04 AM, said:

In a nut shell, yeah. Only in my case it was targeting, my constructs kept destabalising the kinetochore or inactivating due to heterochromatin silencing. I had to find a structure configuration which worked. And even once I found something that worked, I could not eat my cake and see if the project idea worked. I ran out of time

You need to have a long long talk with your supervisor and adviser. These people have more experience in determining if you have enough material for a thesis. And if not, what critical experiment you must do to finish the story.

Much of a thesis is how you defend your work, why you did what you did. Your objectives and how you designed your experiments to get there. Your conclusions and the evidence in your datum to support said conclusion.

And a lot of editing.