Ok so about two weeks ago I think you can recall that I was missing my Extraction Kit so I began extracting DNA from E.coli using a different protocol. My extraction was  successful, but there was a small amount of it. Anyway, when I finished the extraction Josh asked me to try this new RFLP project in what is now my actual project, on E.coli. This RFLP stands for restriction fragment length polymorphism, and is a technique that basically cuts the DNA sequence at specific spots. Certain enzymes in this case restriction enzymes are the ones who look for the same sequence repeating throughout the entire DNA and pretty much just "break" it, or digests it at that spot.  I check my results for this project by running my DNA in an electrophoresis gel. When I first tried it with the E. coli I had too small of an amount to believe that anything could appear and figure 1 shows this. Below are the amounts of DNA, enzymes, H2O, and buffers that I used. It was not successful and one of the reasons why is because later on Matt and Josh realized the buffer and enzymes I used were about 5-6 years old! Of course this wasn't going to work!


Figure 1
RFLP using E.coli
Ruler, A, B, C

Tube	10x buffer	DNA	H2O	Hind 111	Ecor 1
A	2ul	1ul	17ul	0ul	0ul
B	10ul	50ul	32ul	8ul	0ul
C	2ul	2ul	17ul	0ul	1ul

This week some new enzymes and buffers came in and I was able to try the RFLP technique for the first time. I had to make a few calculations to figure out how much of each (buffer, enzyme, DNA) I would need for a total of 100ul of volume. My conclusions were the following

1. 10ul of 1x buffer
2. 88ul of DNA
3. 2ul of Ecor1 enzyme

I added them in this order as well, because if you add the enzyme before you have everything you want in the test tube than this enzyme will actually start to react with itself and whatever is in the test tube. After added all these reagents together, I placed them in a water bath that was set at 37 degrees C for an hour. After that I followed the electrophoresis gel protocol with 1% agarose.



RFLP
Ruler
S. marcescens (3-38-13)
S. marcescens (3-26-13)
S. aureus (3-38-13)
P. stuartii (4-2-13)

 

1. 1ul sybr green
2. 2ul orange loading dye
3. 8ul of DNA

1. 9ul molecular weight ruler
2. 1ul sybr green

I added them in the order you see. The gel ran for 35 minutes at 120 volts in dark conditions. You can see from my results that there was some digesting going on but not like I was expecting.