pARA-R Restriction Digest

Introduction and Confirmation

Examine the role of restriction enzymes and their importance in genetic engineering and bacterial plasmid revealing how it is used in biotechnology

Restriction Enzymes BamH1 and Hind III are used Will digest recombinant plasmid, pARA-R rfp gene will be isolated from larger fragment which contains ampr, araC and PBAD
Independent Variables: pARA-R that expresses rfp gene, Enzyme Mix:BamH1 and Hind III Dependent Variable: Size of restriction fragments (kbp)

Background
Transformation is the taking up of foreign DN like plasmid into bacteria Restriction Enzymes break bonds in the sugar phosphate back bone of viral DNA into small framgments Either clean cuts or staggard, creates restriction fragments which may have sticky ends that can pair with complementary ends of other ends Typical Functions include cloning gene expression Gel Electrosis: DNA has negative charge and migrates toward positive end Supercoiled is compact molecule which moves quickly, Nicked circle where some stress cased a break will not move as fast Multimer which are linked plasmids will move the slowest

Hypothesis
If the restriction enzymes are applied to pARAR the fragments created and examined through gel electrocis then the bandswill be at the expected marker fragments. DNA bands proudeced by A- will be near 6 because its 3 kbp and the bands produced by A+ between 9 or 10 becase the rfp gene is .720 kbp

Methodology Materials:
Reagents:
pARA(70 ng/ ul) Restrriction enzymes ( BamH I + Hind III) 2.5x restrictoin buffer Distilled water (dH2O) Plasimid samples: A- and A+ .8 % agarose gel 1x SB (or .5x TBE) DNA size marker(25 ng/uL)

Equipment and Supplies:

P-20 micropipette and tips 1.5 mL microfuge tubes Mincenterfuge 37 C water bath Permant marker Electrophoresis apparatus Power Supply Plastic microfuge tube rack

Procedure(2a)
1. Obtain three 1.5 mL microfuge tubes : pARA-R, enzyme mix and 2.5x restriction buffer. 2. Obtain two clean 1.5 mL microfuge tubes and use a marker to label the tubes as follows: A+ and A-. 3. Use a fresh tip and add 4L of 2.5x restriction buffer to both tubes. 4. Add 2L of dH2O to tube labeled A-. 5. Use a fresh tip and add 4L of pARA-R to tubes labeled A+ and A-. 6. Carefully add 2L of the enzyme to A+ tube ,mix directly into the solution in tube A+ containing plasmid and buffer. After the addition of the enzymes, cap the tube and gently flick the lower portion of each tube to mix the contents. 7. If there is a minifuge available, set the tubes into the rotor, being certain the tubes are in a balanced configuration, and spin the tubes for four seconds. This 8. brief spin will pool all of the reagents at the bottom of each tube. 9. Place both tubes into the 37C water bath, and incubate for at least 60 minutes. 10. Following the 60-minute incubation, place the tubes into the 11. freezer until you are ready for electrophoresis (Lab 4a).

Procedure (4a)
1. Collect both plasmid samples and the DNA marker 2. Add 2 L of loading dye to the A+ and A- tubes. Do not to contaminate the plasmid samples. Without creating bubbles, gently pump the pipette several times to mix the loading dye with the plasmid samples. Prepare the gel and electrophoresis chamber to receive these plasmid samples. 3. Be certain the gel wells are oriented closest to the negative (black) electrode. 4. Pour the 1x SB (or 0.5x TBE) buffer into the electrophoresis box until there are no dimples. 5. Load your samples into the gel

Using a clean tip and set your P-20 micropipette to 10 L. Aspirate 10 L of your DNA size marker and slowly dispense it into the well. Using a clean pipette tip, load 12 L of your A- sample into the adjacent well. Change the pipette tip and load 12 L of your A+ sample into the next well. Close the gel box lid tightly over the electrophoresis chamber. Connect the electrical leads to the power supply. Be certain that both leads are connected to the same channel (same side) with the negative (black) to negative (black) and positive (red) to positive (red). On the power supply, set the voltage to 130-135 v. Observe the dye movements