[Hplusroadmap] First call for kit additions

[Bryan Bishop]

The following is an exceptionally, inexcusably brief portion of text 
that I found myself writing a few days ago to serve as a simple 
introduction into the basics of DNA restriction cuts and synthesizing 
customize strands. I welcome any comments and revisions before I throw 
it into the svn for the next release (btw, hosting is moving to 
Sourceforge).

   The first step of inserting new genes into an organism is the DNA 
preparation phase. This is where you use an oligonucleotide machine 
with the inkjet cartridges to make the 100 to 200 bp strand of DNA that 
you want to make. The total possibilities for strands is something like 
4^200, but only a subset is interesting and useful. Anyway, in the DNA 
preparation phase you make your base pair (bp) sequence. Store this. 
Make lots of copies. Most transfection/transformation protocols call 
for up to 100 micrograms of the new DNA, and it’s generally hard to 
mass produce large volumes of DNA (can somebody solve this please?).  

Most protocols call for the preparation of DNA before you introduce it 
into your cell culture. I don’t know if this is supposed to mean that 
you should have a complete strand of DNA, or if there’s something else 
going on here, but I think I can make a case that the DNA has to be a 
complete strand. Before the transfection/transformation experiment, I 
suspect that researchers are making their oligonucleotide DNA with 
their specialized base pair sequence, and then using some sort of 
polymerase chain reaction to amplify the amount of constructed DNA. 
Then, they use the dice and splice method with restriction enzymes to 
cut up strands of extracted DNA from the organism that they are 
interested in. Then, they inject their constructed DNA with certain 
starts and tails that are able to latch on to the cut sites of the cut 
DNA, thereby closing the gap. There are a certain number of DNA 
molecules that you are going to have, and a certain number of 
constructed DNA molecules, so I also suspect that with proper mixing 
you can have a one-to-one correspondence and match up, but this might 
not be necessary because the transfection/transformation protocol 
probably calls for more DNA than could be injected into the cell 
colonies.  

You will need to select a “transfection/transformation control.” How do 
you know if your synthetic DNA has been inserted? One really hard way 
is to test the proteins that the cell colonies produce, by doing lots 
of analysis with instruments. Another method is to tap into the DNA 
preparation phase and instead of only adding your new genes, you also 
add some gene that causes the production of a fluorescent protein so 
that you can literally see it glow and you can see which colonies 
(you’ll get colonies at the end of the transfection/transformation 
protocol) actually received the new genetic material. 

“One of the cheapest (and least reliable) methods is transfection by 
calcium phosphate, originally discovered by S. Bacchetti and F. L. 
Graham in 1977.[1] HEPES-buffered saline solution (HeBS) containing 
phosphate ions is combined with a calcium chloride solution containing 
the DNA to be transfected. When the two are combined, a fine 
precipitate of the positively charged calcium and the negatively 
charged phosphate will form, binding the DNA to be transfected on its 
surface. The suspension of the precipitate is then added to the cells 
to be transfected (usually a cell culture grown in a monolayer). By a 
process not entirely understood, the cells take up some of the 
precipitate, and with it, the DNA.”  

- Bryan
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Bryan Bishop
http://heybryan.org/