Title: Size Analysis of Recombinant Plasmids Isolated by a Rapid Miniprep Procedure

Aim: To confirm biochemically that the transformants created in experiment 10 contain

plasmid DNA, and that plasmid size is consistent with that expected, which is determined by
agarose gel electrophoresis.
Abstract: The completion of a series of few simple steps resulted in the isolation of plasmid
DNA from cellular proteins, lipids, and plasmid DNA. The Rapid Miniprep method being the
procedure of choice employed.3It was chosen because plasmid DNA needed to be isolated from
the cell and this method is a quick and very efficient one. Entry was made into the bacterial cell
using lysozyme after which the cell contents (proteins, lipids) were dissolved using SDS and
plasmid DNA denatured using NaOH. Further treatment with ammonium acetate, isopropanol,
ethanol and a solution of TRIS-HCL, pH8.0, 1mM EDTA, 10ug/ml RNase resulted in the
isolation of plasmid DNA.3After completion of the experiment an electrophoretic diagram was
obtained that proved that the plasmid was not isolated successfully. Hence, the experiment can be
considered unsuccessful on the experimenters part.Therefore, reference was made to another
sample. When a plasmid is isolated and an electrophoretic analysis is carried out various
topological forms of DNA is observed; they are the supercoiled, linear and nicked/relaxed
conformations. Two topological forms were observed for the sample (linear and supercoiled);
only a supercoiled conformation of the recombinant (pTrc99A cysB) was observed whereas both
linear and supercoiled topologies of the vector (pTrc99A) were observed. The vector has an
expected size of 4176bp and an actual size of approximately 3200bp whereas the recombinant
had an expected size of 7978bp and an actual size of over 10000bp.3Therefore, it is quite evident
that the plasmid extracted was the vector.

Introduction: The mini-preparation is a simple, however, efficient method of isolating

plasmid DNA from the cell. It utilizes various chemicals at each step in the procedure to obtain a
desired result. The first step in the procedure involved theuse of glucoseand the chemicals TrisHCL and EDTA. Glucose acts to maintain osmotic pressure and the Tris buffers the cell at a pH
of 8.0. EDTA binds to divalent metal cations in the lipid bilayer, which weakens the cell
envelope.1The next chemicals that are used are NaOH and SDS. NaoH causes cell lysis whereas
SDS detergent dissolves the lipid components of the cell membrane and cellular proteins.
Sodium hydroxide also denatures both the chromosomal andplasmid DNA into single strands;
the two strands of intact plasmid DNA remainintertwined. Ammonium acetate is then added to
bring the pH to neutrality and the DNA strands can renature. The large chromosomal strands
cannot rehybridize perfectly though, however, instead they become a partially-hybridized tangle.
Ammonium acetate precipitates the SDS (with its lipids and proteins) from the solution. The
SDS/lipid/protein precipitate traps the tangled chromosomal DNA.1This creates the white goop
that pellets have after centrifugation.1 Only the plasmid DNA, small fragments of chromosomal

DNA and RNA remain in solution. Isopropanol which is added next rapidly precipitates nucleic
acids. However, if allowed to sit for longer, proteins will also precipitate. The DNA is then
washed with ethanol. An ethanol wash helps remove salts and any remaining SDS as these can
interfere with a restriction digest. The final step in the procedure which is to suspend the DNA
into Tris-HCL and EDTA is done because Tris buffers the DNA solution.1 EDTA binds divalent
cations (especially Mg++ ions) that are a needed cofactor for bacterial nucleases and thus limits
DNA degradation. After isolation of the plasmid occurs it is analyzed by gel electrophoresis.
Agarose gel electrophoresis is a method of separating DNA fragments based on size and being
able to view them.2This technique is based on the knowledge that DNA is negatively charged at
neutral pH to its phosphate backbone. Because of this fact when an electric potential is placed on
the DNA it will migrate towards the positive pole. It is also used to give the sizes of DNA
fragments after the procedure is complete.2

Method: As seen in Experiments in Molecular Biology: Biochemical Applications, Pages

137-139.
Changes made to the procedure:
1. In step 9 the sample was centrifuged for 10mins instead of 5mins and at room
temperature instead of in the cold room.
2. In step 15 the DNA pellets were suspended in 20ul of Tris-HCL instead of 50ul.

Discussion: Plasmids are circularized strands of DNA found in a bacterial cell that is
separate from the chromosomal DNA that is present and replicates independently of the host's
chromosomal DNA. A recombinant plasmid is a plasmid that has been cleaved at a specific site
and a DNA sequence is introduced into the cleavage site to form a recombinant
plasmid/dna.Plasmids can be easily isolated from the bacterial cell using a method such as the
Rapid Miniprep procedure. In order to be sure that successful isolation of the plasmid DNA
was accomplished the plasmid was run on an electrophoretic gel. After running the plasmid on
the gel an electrophoretogram was obtained. This indicated whether or not any plasmid was
isolated and if so the size and topological forms of the plasmid.Thus,it gives an indication of
whether or not the experiment was successful.

There are three topological forms of DNA, namelysupercoiled, nicked and linear. These three
topologies are of different sizes and accounts for why one fragment of the DNA travels further
than the other. The supercoiled DNA is the fastest moving topological form of dna of the uncut
plasmid. The supercoiled DNA has a very compact structure and for this reason is the fastest
moving conformation in the gel. This is because the agarose gel is of a matrix form hence the dna
has to move through the matrix. For this reason it is safe to say that the band that travelled the
farthest was the supercoiled conformation. Another conformation of DNA is the nicked (relaxed)
conformation. This may occur when topoisomerase nick on strand of the DNA helix so that DNA
polymerase has access to DNA for replication. Once this happen the super helical tension relaxes
and tightly-wound ball becomes a floppy circle. A nick may also occur during the isolation of the
plasmid because of mechanical shearing of the DNA. For this reason the nicked circle is the
slowest conformation of uncut DNA. The last conformation of DNA is linear DNA which is
produced when a restriction enzyme cuts a plasmid only at one site. It can also occur because of
endonuclease contamination of the isolated plasmid, or because of mild treatment. The linear
DNA will run between the supercoiled and nicked conformations on a gel (possibly closer to the
supercoiled band).
On observation of the electrophoretogram obtained it is quite evident that two bands were
obtained. However, the two bands obtained were very faint in colour. Also they barely moved
down the gel. Thus, it is quite obvious that this result cannot be usedbecause it was not done
properly; a variety of reasons may be the cause of this result obtained. It may have to do with the
concentration of dna loaded. An insufficient quantity of the concentration of the dna being loaded
on the gel might have been the cause.4 Increasing the amount of dna or ensuring that the correct
volume of dna was taken up might solve this problem. Another reason is that the dna might have
been degraded by nucleases.4 More care should be taken when carrying out the experiment to
avoid contamination of the dna (avoid touching everything that contact is made with). Smearing
of the dna was quite evident on the electrophoretogram obtained. Various factors may cause
smearing to occur. It may be because too much dna was loaded on the gel. Decreasing the
amount of DNA used might solve this problem.4Again, it may be that the DNA has been
degraded by nucleases. Another reason that causes smearing to occur is that of contamination
due to protein.4 This problem can be solved by ensuring that after adding isopropanol the
solution is not allowed to stay for too longer before moving on to the next step as this will also
cause precipitation of the protein to occur.
The fact that the result obtained was unsuccessful meant that reference had to be made to a
successful one. Therefore, reference was made to lane A8 sample B. In this lane two topological
forms of DNA were observed; they were supercoiled and linear. In order to determine which
plasmid was obtained, the size of this plasmid was compared to the size of the vector (pTrc99A)
and recombinant plasmid (pTrcp99A topA-cysB).Only one form of the recombinant was clearly
visible-this was considered to be of the linear topological form based on the distance moved.
However, there were two visible topological forms of the vector even though three bands were

seen. The bands were of the linear and supercoiled form. The one (vector or recombinant) that
most closely matches in size to the sample meansthat it was that plasmid that was obtained. The
expected size of the vector was 4176bp and that of the recombinant plasmid was 7978bp3.
However, these were not the experimental sizes obtained. The size obtained for the vector was
approximately 3200bp and that for the recombinant was above a little above the 10kb ladder. The
fact that the expected size of the vector was 4176bp and the size obtained was 3200bp means that
the plasmid might have been smaller than one thought and so would move a greater distance
down the gel in its supercoiled conformation than one would expect. However, on the other
hand, for the recombinant with an expected value of 7978bp and an experimental value greater
than 10000bp means that the plasmid did not move as far down the gel as one would expect. For
the sample that was run also, an approximate size of 3300bp was obtained. Based on all the sizes
obtained it is quite evident that the plasmid obtained is the vector-pTrc99A (3200bp vs. 3300bp).
Limitations that may have aroused in this experiment may include

References:
1. http://www.ppsk.usm.my/lecturers/mravi/PDF_FIles/Plasmidextraction2002.pdf
Date of retrieval: 24/09/2013
2. http://faculty.plattsburgh.edu/donald.slish/Electrophoresis.html
Date of retrieval: 24/09/13
3. Zachary F. Burton. (1997).Experiments in Molecular Biology: Biochemical Application,
Size of pTrc99A and pTrc99A cysB pages 48 and 143. Experiment 12A pages 137-139.

4. http://bio.classes.ucsc.edu/bio20L/info/content/molbio2/molbio1/troub.htm
Date of retrieval: 24/09/13

Questions:
1. Did you obtain recombinants of pTrc99A containing the topA gene? What evidence do
you have to support your conclusion?
No recombinants of pTrc99A containing the topA gene were obtained. First of all because no
bands were obtained on the electrophoretogram and the sample that was referred to, although
having DNA fragments of different topologies; none of the topologies matched the recombinant
plasmid in size. They were instead of similar size to the vector; it was therefore, concluded that
no recombinants of pTrc99A containing the topA gene was obtained.

2. Do the transformants of XL-1Blue all contain the topA-cysB fragment in pTrc99A?

The transformants of XL-1Blue did not all contain the topA-cysB fragment in pTrc99A because
the fragments obtained werent aligned with the control. If the fragments had the gene then they
would be of similar size to that of the control and would have moved similar distances on the gel.
Hence it is safe to say that the XL-1Blue transformants did not have the topA-cysB fragment in
pTrc99A.

I will make a guess that you are asking about the restriction enzyme buffer. Take one step back.
When evaluating DNA, it is common to use a restriction endonuclease which makes very
specific cuts along specific palindromic sequences on the DNA. The restriction digested DNA is
then subject to gel electrophoresis.
The restriction endonucleases are enzymes and like all enzymes has an optimum temperature and
salt concentration at which they work. Restriction enzyme buffers have the appropriate salts and
sometimes include essential co-factors that allow the enzyme to function. If you weren't to use
the appropriate buffer, chances are the enzyme didn't work, the DNA didn't cut appropriately and
your conclusions about the DNA you use will be wrong.

When working with DNA samples, you'll often need to digest them with restriction enzymes,
meaning you'll add restriction enzymes to make cuts in the DNA. The kind of restriction
enzymes you'll typically use are type II restriction enzymes, which make cuts at specific sites.
These restriction enzymes will be added together with a restriction buffer, which contains
ingredients that ensure the restriction enzyme will work properly.

Read more: http://www.ehow.com/info_8506104_function-restrictionbuffers.html#ixzz2gHSjil3l