Lab 8&9x

disgustedtukwilaInternet and Web Development

Dec 14, 2013 (3 years and 6 months ago)

76 views











Joshua Sadaj











BioChem 380
-
03











October 25, 2010


Experiment 8/9: Plasmid DNA Isolation, Restrictive Enzyme Digest & Electrophoresis of DNA


Introduction


The purpose of this lab was to first isolate plasmid DNA from E. coli cell by using a procedure called
alkaline
-
lysis miniprep. In short, this technique separates plasmid from genomic DNA and cellular
structure. The bacterial cell is then lysed by degrad
ing the cellular wall. A treatment with SDS
denatures proteins and disrupts cellular membranes. Precipitating the cellular contents with a high salt
solution will leave the plasmid DNA in the supernatant and the rest of the
cell and its pieces will remai
n
on the bottom. The purpose of the second part of this lab was to use gel analysis to analyze the plasmid
DNA from part I by using a restrictive endonuclease, which will cut the plasmid DNA and different
recognition sites. The third part of this lab was

to determine the sizes of the DNA samples by way of
electrophoresis. The larger pieces of DNA will travel much slower than the smaller pieces yielding a
much shorter distance through the gel. Measuring theses distances will show the size of each sample,

therefore the longer the distance traveled the smaller the DNA sample and the opposite for the larger
pieces they will only travel a short distance.


R
esults:








1
10
100
1000
10000
0
0.5
1
1.5
2
2.5
3
3.5
Size (bp)

Distance traveled

DNA Ladder

Ladder
DNA Ladder

Band

PUC Ecoli

pUC18

JS

AW

JC

TAQ

Ladder

1

2.8

2

.6

.2

.6

3.6

.2

2



1

.8

1.3

4.75

1.55

3



1.3

1.1

1.55

5.6

1.8

4



1.55

1.35

2.2


2.05

5



2.25

2.35

2.55


2.3

6



2.6

2.2

2.3


3.05

Table 1: Results from electrophoresis in inches


Band

pUC Ecoli

pUC18

JS

AW

JC

AW

Ladder

1

1271.02

2071.39

6474

8965

6474

8965

6557

2



4674

7542

3662


4361

3



3662

6443

2987



4



2987

4997

1760


2322

5



1689

3567

1271


2027

6



1271

2246

1546


564

Table 2: Estimated base pairs, per band on gel


Discussion:



Part I was to strictly isolate the plasmid DNA from the rest of the cell, which was doen
using a series of different washes. The end result was plasmid DNA left in the
supernatant that could be separated from the rest of the material.



Part II was where th
e plasmid DNA was cut using restrictive enzymes that recognize
certain DNA sequences in genetic code. Taq
-
1 was used to digest the pUC18 plasmid.
Taq
-
1 binds the sequence 5’
-
TCGA
-
3’ and will cut the plasmid at a minimum of two
points. These cut strands
can then be separated by size on ag
ag
rose gel, which was
done in part III.



The cut DNA was separated in an ag
ag
ro
se gel. The larger pieces of DNA (more base
pairs) have a more difficult time moving through the gel and therefore do not move as
far.
Smaller pieces move much more easily in the gel and therefore travel further in a
given amount of time. The uncut DNA doesn’t travel as far as well due to its size and
large number of base pairs.



In part III a DNA ladder was used to obtain a graph of mole
cular weight vs. distance
traveled. This graph was then used to extrapolate the molecular weights of the cut and
uncut control samples by using the distance traveled. When a trendline is created for
the DNA ladder, the molecular weights of the uncut and
cut data just do not seem to fit
what is known about the pUC18 plasmid DNA.



The size of the picture taken of the gel does not represent accurate size and the
measurements of distance traveled are therefore skewed. If the distance traveled is off,
then the

molecular weight is also going to be off since distance traveled was used to
extrapolate molecular weight.



Part III yielde
d a different answer for A. W
’s cut plasmid because the proper amount
required for the well was not added, some was lost in partI.


Q
uestions:


Page 57

1.

pUC 18 plasmid is form E. coli bacterium and is 2600 base pairs in six and specifically codes for
antibiotic resistance to Ampicillin.

2.

So we know we are getting the pUC18 plasmid since there can be many plasmids in a bacterium.
When we
add the culture to Ampicillin, only the E. coli with pUC18 gene sill survive.

3.

A

4.

A

5.

A ligase is an anzyme that catalyzes the joining, or ligation of two substates.

6.

A restrictive enzyme cuts DNA strands at specific sequences.

7.

Taq
-
1 is purified from a strand o
f E. coli that carries the Taq
-
1 overproducing plasmid pFBLT88.

8.

Taq
-
1 is a restrictive endonuclease which is an enzyme that cuts nucleic acid at specific
restriction sites and produced restrictive fragments. Taq
-
1 specifically recognizes certain DNA
seque
nces on the pUC18 plasmid and can make up to four different cuts in the DNA. In this case
it recognizes a palindrome sequence in the DNA.

9.

A palindrome is a sequence that can be read the same was in either direction.


Page 61
-
62


1.

See results

2.

The restriction endonuclesase cuts the plasmid at four different sites creating three different
sizes of plasmid DNA.

3.

The recognition endonuclease will function the same as it does on any DNA strand and will
recognize a certain sequence on the double stran
ded DNA, and cut it at this point T/CGA.

4.

The restrictive endonuclease will cut the plasmid and create 3 separate fragments.

5.

There would be only on DNA segment with a large number of base pairs causing it to only travel
a short distance through the gel.

6.

Se
e picture.

7.

Ethium bromide is added to the agagrose gel so the bands can be seen. This dye is a mutagen
that does not interact with the agagrose gel but intercalates between tha aromatic rings of the
bases of DNA strands.

8.

High voltage can cause the agragos
e gel to melt, so low voltages will yield better resolution.
Longer run times will allow the bands to separate more so. Bands which are around the same
molecular weight will be more separated from each other with longer run times.

9.

RNAse

catalyzes the hyd
rolysis of ribonucleic acid. It degrades RNA into smaller fragments.
Adding DNAse to the sample would catalyze the phosphodiester bonds of the DNA backbone.
Depending on the DNAse added, it can cut DNA at specific sites. We used Taq
-
1 for that
purpose.

10.

The DNA ladder was used to create a graph of molecular weight vs. distance traveled through
the agagrose gel. This graph was then used to extrapolate the molecular weights of the cut and
uncut plasmid DNA by using the distance that their respective bands

traveled through the gel.

11.

&12. Prokaryotes contain two different types of DNA: plasmid and chromosomal. Whereas
Eukaryotic cells carry only chromosomal. Prokaryotes posses DNA that is circular there the DNA
in Eukaryotes is linear. Eukaryoti
c DNA is also much larger than Prokaryotic DNA.

12.

The misuse of antibiotics has led to antibiotic

resistant bacteria. By prescribing antibiotics in
cases not necessary, we are ultimately selecting for bacteria that are resistant to that drug. The
drug will

kill off any bacteria which do not contain the antibiotic resistant plasmid, thus leaving
only the resistant bacteria to reproduce and multiply. Antibiotics should not be used unless
they are necessary.

13.

Plasmids can convey antibacterial resistance to hos
t cell and therefore can be very useful to
scientists studying bacteria and what they are resistant to in order to help treatment of bacteria.
Plasmids are also useful in gene therapy and are currently being researched.