Dieu Truong
BTECH 1015
12/5/2013
Identified plasmid by restriction enzymes
Introduction:
Chromosomes usually have a circular shape. Plasmid are inserted a circular pieces of
DNA, it smaller than chromosomes. Plasmid replicate independently from the chromosome.
Plasmids are used to multiply the gene of interest and to transfer foreign genes into the cell by
cell transformation process. Cell may have 1 copy of plasmid or many up to 100. It is
importance of antibiotic when culturing bacteria carrying plasmids. Restriction enzyme
(restriction digest) are enzymes that cut DNA of a particular sequence. Double digest have 2
restriction are added. The single and double digest used to identify a plasmid. All restriction
digest must both work in the same buffer. Gel electrophoresis can be used to separate DNA,
RNA and proteins. The separation based on the movement in an electric field. When the
restriction digest cut DNA to apart, and run over gel, the short DNA fragments move faster than
the long DNA fragments. DNA fragment after run over gel will show the travel distant on the
gel. It will help to determine what the plasmids in the cell. When the plasmid in the cell was
identified, it helps to understand the purpose of the plasmids. To achieve this goal I have to
have a skill of set up the restriction and run DNA over gel.
Methods:
I received the unknown plasmid from instructor. I used the NanoDrop to find my
concentration. Loaded 2 uL of the plasmid, checked the loading and then closed the arm,
measured the concentration of the plasmid. My concentration result was 146.6 ng.
Concentration helped me calculate the amount of the plasmid that I need to put on each of
three 1.5 mL microfuge tubes. I used the PstI and EcoRI-BgII as my single digest and double
digest. All of those digest work in the buffer O and I got it from the cold room of school
laboratory. Each of three 1.5 mL microfuge tubes was included the unknown plasmid, 10x
buffer O, one of those microfuge tubes had PstI enzyme, was labeled by “P”, another tubes
labeled by “E+B” contained EcoRI enzyme and BgII enzyme, the last tube labeled by “Control”.
Placed the tubes in the 37°C water bath, and incubated overnight.
I made 0.8 % agarose gel. I measured out 0.32g agarose , then poured into the small
glass bottle and added 40 mL of 1x TAE, placed the lid on the bottle. After that, I mixed the
solution by put it into microware for 1 minute for make sure all of the agarose is totally melted.
1 uL of the ethidium bromide solution was added in to the melted gel mixture. I waited several
minutes for the melted solution get cool, then poured it into the gel tray which closed each
open of the gel tray with lab tape and waited 20 minutes for the agarose to solidify.
I made 200 mL of 20x TAE buffer to run the gel. 20x TAE included 800 mM Tris-acetate
(Tris base +acetic acid) I calculated amount of Tris base) and 20mM EDTA. I calculated amount
of Tris base by solved the problem:
0.8 M/L * 0.2L*121.14 g/mol =19.3824 g
And I calculated amount of 0.5 M EDTA (pH 8) by solved the problem:
(0.2 L*0.2 M)/0.5 M= 0.08 L = 8 mL
I dissolve 19.3824g of Tris base into the beaker had 150 mL of water and added 4.6 mL glacial
acetic acid in to the same beaker and mixed it together. 8 mL of 0.5 M EDTA was added in to
the beaker and mixed. By using a calibrated pH meter, I determined the pH of my solution was
8.2, and then added water to bring the final volume to 200 mL. I took my three tubes of
plasmids out, added 4 uL of 6x orange loading dye to each tube, mixed by pipetting up and
down. After 20 minutes the gel was solidified, I removed the gel comb and the tape from the
ends of the tray, and placed the gel and tray into the gel box, with the walls of the gel tray
touching the sides of the gel box. I filled the gel box with the 200 mL of 20x TAE buffer until the
gel was fully covered with buffer. I loaded the gel with my plasmids from three tubes and
ladder DNA from laboratory like an order below and ran it at 130 V for 45 minutes.
6 uL ladder/ blank / 20 uL control / 20 uL P / 20 uL E+B/ Blank
After 45 minutes, until the lower dye band has migrated to half inch from the bottom of the gel,
I took the gel out of the gel box, and recorded images of gel by take a picture on the UV
imaging system. From the picture, DNA fragments moved further have a smaller size.
Therefore, base on the traveled distance of the DNA fragment I can determine the size of it.
I used the NEB Cutter tool (http://tools.neb.com/NEBcutter2/), a tool provided by New
England Biolabs, to get the fragment sizes (bp) for pAMP, pKAN, and pBLU from the digestion
with PstI enzyme and double digest EcoRI-BgII. I also used the software called Microsoft Excel
2010 to make the Standard Curve to determine the sizes of the DNA fragments in the unknown
plasmid.
Results and Conclusions:
Tube
Miniprep
plasmid
(350 ng)
10x buffer
PstI
EcoRI
BgII
dH2O
Total
volume
Control
3.4 uL
2uL
---------
---------
--------
14.6 uL
20 uL
P
3.4 uL
2uL
1 uL
---------
--------
13.6 uL
20 uL
E+B
3.4 uL
2uL
----------
1 uL
1 uL
12.6 uL
20 uL
O
Table 1: The recipes of restriction digest base on the concentration of the unknown plasmid.
Figures 1: images of gel with a lanes order like: ladder/blank/control/P/E+B/blank. P lane has
4556bp, E+B lane has 2 bands was showed up: 3200 bp and 1118 b.
DNA fragment sizes (bp) produce from digestion with
Enzyme PstI
Enzymes EcoRI and BgII
pAMP
4556
6,152,1118,3200
pKAN
3271,923
109,152,794,3155
pBLU
3924,1316,197
2121,145,1431,1756
Experimental plasmid
4700
1200, 3300
Table 2: Fragment size for pAMP, pKAN,pBLU predicted by the NEBcutter tool, and experimental
data obtained from the standard curve in figure 2.
Figures 2: Standard curve graphs, with equation y=-0.88ln(x)+9.3167 and the R2value is 0.9719.
the line properly fits the data, the graph showed PstI digest cut the plasmid at 4700bp (data was
4556bp), B+E double digest cut plasmid at 1200bp (data was 1118bp) and 3300bp (data was
3200).
Base on the experimental data obtains from the standard curve (figures 2), I saw my
plasmid was cut by PstI digest has the fragment size is 4700bp, it’s near with pAMP of the
predicted by NEBcutter tool: 4555bp. I also saw my plasmid was cut by EcoRI+BgII double digest
has two fragment, the size was 1200bp and 3300bp, while the predicted was 1118bp and
3200bp. The predicted results also show a fragment size of 6bp and 152bp but those fragments
are too small to be seen on the gel or compared with the ladder. Thus, both my single and
double digest results strongly suggest my unknown plasmid was pAMP.