Lab report

1. Genetics Lab Report 1
GENETICS LAB REPORT
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2. Genetics Lab Report 2
Introduction
The process of construction of a recombinant plasmid in DNA size determination
involves a connection of the insert yeast DNA in the backbone of a compatible vector (Brown,
2010). The process of connection is facilitated by the sugar backbone of the two fragments of
DNA. The method is used to move pieces of DNA through a variety of vectors after binding the
restriction sites. The technique is a common laboratory practice that is also widely used to move
markers, promoters as well as other DNA elements between plasmids (Lee, Costumbrado, &
Hsu, 2012).
Aim/Objectives
The primary aim of the practical is to establish the size of a yeast DNA that is cloned into
a vector pCK103. The practical also entails the establishment of a primary restriction map.
Requirements
The following materials are incorporated in a 1.5ml tube before conducting the experiment:
 1μg DNA
 3μL 10x Buffer
 3μL 10x BSA
 1μL Restriction enzyme
 Distilled water
The reaction buffer selected should be compatible with the restriction enzyme selected in the
experiment. The restriction enzyme used should be placed in on ice after it is removed from -
20°C freezer (Brown, 2010). Exposure of the enzyme to heat can easily cause denaturation and
subsequent loss of function. Also, the amount of restriction enzymes used is dependent on the

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quantity of DNA required for cutting. 1μg of DNA in a 50 μL is derived from one unit of
enzyme in a one-hour reaction (Surzycki, 2012).
Methodology
Question 1
The method demonstrated used in the experiment illustrated in figure 1 is the restriction
digest procedure. The experiment is used for cleaving DNA molecules at specific sites (Surzycki,
2012). DNA fragments containing a similar sequence have equal sizes. In the experiment, unique
DNA-cleaving enzymes are used for cleaving the DNA molecule known as restriction
endonucleases.
The contents of the tube are gently mixed by pipetting and later incubated at a temperature of
37°C for 1 hour or as instructed by the kit manufacturer (Surzycki, 2012).
After the digest, the DNA is later amplified using PCR. The process of amplification is to make
the DNA be easily analysed using agarose gel electrophoresis technique. In this method, the
proteins are separated depending on the size and charges. Agarose gel is commonly used because
they are easy to cast and are efficient in the separation of DNA of different sizes (Surzycki,
2012).
Results
Question 2
During the process of loading and running the samples, both positive and negative
controls are included. A standard sample with fragments of known sizes is also used. The
positive control sample usually contains the known DNA sample and hence the gene required
(Brown, 2010). A negative control sample does not contain any genetic material.

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In the experiment, Lane 1 is the standard ladder. The lane contains many fragments of
pre-determined sizes and is mainly useful in for comparison purposes for the other lanes. Lanes 2
and 3 are the negative controls. The two Lanes have uncut pCK103 and Pham93 respectively,
and they do not contain any enzymes (Brown, 2010). The uncut vector in lane 2 is important for
checking the viability of the competent cells. The control is also important in demonstrating the
antibiotic resistance of the plasmids.
Lane 7 is the positive control. The lane contains pCK103 cut with EcoRI. The enzyme
has a size of 3118 base pairs which is almost the same size as illustrated by the standard.
Therefore, lane 7 has the required gene in the sample.
Discussion
Question 3
Gel analysis is used to demonstrate the sizes of the DNA in base pairs. The results are
based on the standard used. In the experiment, agarose gel electrophoresis was the preferred
method of analysis. After loading the samples and the controls, the gel facilitated the movement
of the DNA strands along the medium. The migration of the nucleic acid was determined by the
size of the DNA, the dimension of the gel pore and the voltage used in the process of
electrophoresis. Other factors that are a determinant in the results of the experiment include the
concentration of the intercalating dye as well as the ionic strength of the buffer (Surzycki, 2012).
Separated DNA fragments are stained using ethidium bromide that intercalates between
the major grooves of the DNA strands. Ethidium bromide also fluoresces under the UV rays
hence easily visualized. SYBR Green and Methylene Blue may also be used as alternative stains
(Lee, Costumbrado, & Hsu, 2012).
Question 4

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pCK103 has a size of 3118 base pairs as illustrated in figure 2. The enzyme used in Lane
7 (EcoRI) digested the plasmid once. Since plasmids are circular, cutting the plasmid gives one
linear fragment as illustrated by Lane7. 3530 base pairs is the approximate size of the original
plasmid. However, the size of the plasmid demonstrated by Lane 7 is 3118. Therefore, the results
of the experiment were not very accurate since a single cut of the plasmid should give the
maximum size of the original plasmid.
Question 5
Lane 3 illustrates the uncut pHAM93. The total size of the plasmid can be determined by
adding 9133+ 8236 +3582 =20951. 20951 is approximately 21226 which is the original size as
illustrated by the standard. In Lane 4, pHAM93 was cut by EcoRI twice (3499 + 2543 = 7043).
In Lane 6, pHAM93 was cut twice by Xbal (3047 + 2720 = 5767). In Lane 8, pHAM93 was cut
thrice by both Xbal and EcoRI (2978+ 1912 + 289 = 5179).
Therefore, the size of the yeast insert in pHAM93 is 20952- (7043+ 5767 + 5179) = 2961
base pairs.
Question 6
Simple Restriction Map for pHAM93
Positions of the test Enzymes and insert enzyme along vector pCK103
0 bp 3753bp
EcoRI and HindIII
3499bp2978bp
EcoRI Xbal Xbal
3047bp
pCK103 Insert
2961bp

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Conclusion
In gel agarose electrophoresis, sometimes the results of the analysis may fail to be clear
due to smearing. Smearing is when there are bands tightly together such that it is impossible to
distinguish them. Smearing could result from contaminants on some of the stray nuclease that
results in degradation of the DNA into smaller fragments (Brown, 2010). Another mistake that is
common in the set-up is the use of a wrong buffer, failure to monitor the temperatures and other
bad conditions during the experiment. Conditions that are not conducive to the experiment
results in degradation of the enzymes. The enzymes may also cleave the plasmids at multiple
random sites. The process of electrophoresis is performed in buffered media so that the PH
changes resulting from an electric field can be regulated.
Agarose gel electrophoresis is applicable in many instances that require DNA expertise. For
example, it is used in the analysis of products of PCR in molecular genetic diagnosis as well as
fingerprinting (Surzycki, 2012).

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References
Brown, T. (2010). Gene cloning and DNA analysis: an introduction. Oxford; Hoboken, NJ:
Wiley-Blackwell.
Lee, P., Costumbrado, J., & Hsu, C. (2012). Agarose gel electrophoresis for the separation of
DNA fragments. Journal of visualized experiments: JoVE(62), 1-5.
Surzycki, S. (2012). Basic Techniques in Molecular Biology. Berlin, Heidelberg: Springer Berlin
Heidelberg.