Sanger Method - DNA Sequencing

ddNTP

Sanger Method

1. Start off with a single strand of DNA.  Occurs through denaturation of DNA, be being heated.
2. Radioactively labeled primer attached to four copies of single strand DNA.  (to help identify the starting point)
3. Each strand is put into individual test tubes.  Each test tube is filled with an abundant amount of dATP, dGTP, dCTP, dTTP.  
4. In each separate test tube, a small amount of ddNTP is added to each test tube.  One type of ddNTP for each test tube, e.g. ddATP in one, ddGTP in one, etc.  ddNTP stands for dideoxy nucleoside triphosphate.  It has NO oxygens on the carbon chain, and therefore cannot bond with any other dATPs, which terminates the DNA replication.
5. Polymerase 3 is added.
6. DNA replication occurs.  In each test tube, there will be strands where the replication automatically stops as a result of ddNTP.
7. Each individual test tube is loaded into four individual lanes on a gel.  The gel is run through an electrophoresis machine, and the DNA sequence can be read from bottom to the top, because it is radioactively labelled.  

PCR vs. Vector Cloning

Vector Cloning:

• Relatively cheaper compared to PCR
• replicates entire DNA
• requires bacteria and plasmid
• can be used to insert DNA and produce protein
• requires specific R.E. 
• Has practical uses in treating medical disorder (eg. somatropin for stunted growth)

PCR:

• Requires less time
• Higher probability of damage/corruption.  if only one DNA is replicated improperly, every DNA strand in that set will be negatively affected.
• Only replicates DNA, cannot produce protein
• Useful for forensics; PCR only requires one intact DNA strand.
• Medical uses more centred in help with diagnosis (diagnosis of HIV, looking for HIV genome)

Actually Starting on ISU

Got my paint together, got all the pots and soil and etc. together.

Started mixing paint.  And I suddenly ran into a foreseeable but unforseen problem.

Oil based paint does not dilute very well in water.  Not at all, actually.  Oil and water don't mix.  Duh.  Should've thought of that earlier.

So now I'm wondering if I should just go ahead with the environmentally friendly and acrylic paint and go without the last one, oil based, or find a substitute.  What a dilemma.

I decided to start another blog to keep track of my ISU progress.  Just cause I like blogs.  And cause I'm tired of putting together work logs for my ISUs (multiple curses upon my grade 11 and 12 physics ISUs...)
http://isuayjcc.blogspot.com/