Nihal Nazeem

Ms. Mathews
STEM 1/2
9/26/17
Polymerase Chain Reaction Lab Write-Up

Purpose:

To use Polymerase Chain Reactions, and gel electrophoresis to trace our locational

origins using Alu repeats in our DNA, and developing lab skills.

Hypothesis:

If PCR and gel electrophoresis are conducted on my DNA, then my Alu repeats should

reveal that I am from India.

Procedure and Materials:

This lab was performed in acccordance with BABECs (Bay Area Biosicence Education

Community) guidelines on the Alu PV92 PCR lab found in the following link:

http://babec.org/wp-content/uploads/2016/12/Alu_Student_Guide_2017.pdf

Data:

Under gel electrophoresis, the 2% agarose gel

was run at 150 Volts for twenty minutes, and
stored using GelRedTM loading dye for 72
hours. Row 1 and 2, under the Ladder
column, have a 100 base-pair ladder. Row 1,
columns A-G; and row 2, columns A-G, have
20 mL of a loading dye solution composed of
a 50 mL of DNA to 10 mL of loading dye
solution. The sample on row 2, column A was
the sample I had worked with, had no orange
bands, which meant no result.
Nihal Nazeem
Ms. Mathews
STEM 1/2
9/26/17
Observations:

After rinsing the saline solution, the solution had foam.

After centrifugation, a pellet was formed and dissolved with shaking of the

microcentrifuge tube

After using the heat block station, the solution had vapor

No discernable reaction, with the eye, was observed when the DNA was added to the

Chelex, Master Mix, and Primer Mix.

Proper pipetting stoppage was used, but some of the solutions had remained for certain

steps.

The pipette was not always put right beneath the surface of the solution.

Analysis:

Due to insufficient data, a mock calculation was done:

Allele Frequencies

*Homozygous/Heterozygous Genotype Amount of Students That

Have That Genotype

Homozygous +/+ 15

Heterozygous +/- 10

Homozygous -/- 12
*Homozygous: Having the same allele, in this case the student inherited the same type of Alu repeat, from both parents. +/+ indicates that both parents gave their
child in their childs Chromosome 16 the Alu repeat showing under the UV results of electrophoresis as an orange band of 715 bp. If this is -/-, then both parents
did not give their child in their childs Chromosome 16, the Alu repeat and shows up as an orange band of 415 bp ; Heterozygous: Having different alleles, in this
case one parent has given the Alu repeat, and one has not, showing two orange bands, one of 415 bp, another of 715 bp, for the child, which would be informed
by their childs Chromosome 16. Since DNA is negatively charged due to its phosphate backbone, gel electrophoresis moves the DNA towards the bottom of the
display as the top of it is positively charged and the bottom is negative, drawing DNA to the negative side. Smaller DNA moves faster down than larger DNA.
The orange bands are visible due to the UV light and dye, the Chelex is used to break up the DNA for the Master Mix to attach by the Primer Mix attaching these
bases ot the template DNA.
Nihal Nazeem
Ms. Mathews
STEM 1/2
9/26/17

Since each person has two number 16 chromosomes, there must be twice as many total

alleles as there are people:

For + Alleles:
2(+Alleles) 2(+Alleles)
student * (15 + 10 + 12) students = student * 37 students = 74 Alleles
2(+ Alleles)
Homozygous +/+ Students
(15) = 30 " + " Alleles

(30 Homozygous " + / + ") + (10 Heterozygous " + / ") = 40 " + " Alleles

Frequency of + Allele:

40 "+" Alleles
74 T otal Alleles
= 54.054 %

For - Alleles:

(24 Homozygous " + / + ") + (10 Heterozygous " + / ") = 34 " + " Alleles

Frequency of - Allele:

7440 "" Alleles 34 "" Alleles

74 T otal Alleles
= 74 T otal Alleles
= 45.945 %

Checksum:

40 "+" Alleles + 34 "" Alleles 74 Alleles

74 Alleles
= 74 Alleles
= 54.054 % + 45.945 % = 100% = 1.00

Genotype Frequencies

Hardy-Weinberg Equilibrium: When the expected frequencies for the genotype matches the

allele frequency, causing an equilibrium showing that the system has no change from the

expected, therefore evolution did not exist in the system .

Variable Assigned Value

40
p + Allele frequency ( 74 )
Nihal Nazeem
Ms. Mathews
STEM 1/2
9/26/17
34
q - Allele frequency ( 74 )
p2 + 2pq + q 2 = 1.0

Variable Genotype Frequency of Homozygous or

Heterozygous

p2 +/+

2pq +/-

q2 -/-

40 2 160
p2 = ( 74 ) = 5476
0.29284.... = 29%

40 34 1360
2pq = ( 74 )( 74 ) = 5476
0.49671... = 50%

34 2 1156
q 2 = ( 74 ) = 5476
0.21110... = 21%

Genotype Frequency for the Amount of Students:

p2 (37 students) = 0.2284...(37) = 10.81 students 11 students

2pq(37 students) = 0.49671...(37) = 18.378 students 18 students

q 2 (37 students) = 0.211110...(37) = 10.81 students 8 students

Since the amount of expected students with these genetic frequencies are different than the data

set of the students who hypothetically do have it, evolution stands, as it disproves the

Hardy-Weinberg Equilibrium.
Nihal Nazeem
Ms. Mathews
STEM 1/2
9/26/17
With the fact that there was insufficient data to determine my genetic origins, my

conclusion to my hypothesis would have to be that my results were inconclusive. This is

because, I had no data to analyze and compare to a genetic database. Some errors and

improvements that this lab could have encountered and undergone could have been the lack of

use of crushed ice during Master Mixing, and Primer Mixing stages, which could have given

inconsistent temperatures to the microcentrifuge tube; the contamination of the pipette tips by

using direct hand contact, without the use of gloves; improper pipetting technique, whether it be

using the wrong pipette, pipette tip, stop, pushing the pipette to a stop after putting it in a

solution, or not pipetting just below the surface of the solution; the lack of using a control to

verify that the results received were as they should have been the lack of labeling and rack

recording of the microcentrifuge tubes, causing some to have confusion about their samples;

putting the Chelex solution into ones DNA solution, causing a pipette to possibly be stuck with

a Chelex bead; and improper gel and comb technique where one could have inserted their

solution too deep into the well, missed the well, or broke the gel well with the pipette. I would

improve the lab by having mandatory observations, labeling, and the use of good sterile

technique. I would also include timely and specific instruction, so that all students can perform

the lab around the same times, without being behind for logistical reasons, and plan the amount

of time and placement of time when conducting this lab. Some further exploration of tracking

genetic origins could include other animals to see geological relatedness of one species and using

this as a comparison point to find out how different organisms evolved, and also use this

technique for other organisms to track their geological origins through genetics.
Nihal Nazeem
Ms. Mathews
STEM 1/2
9/26/17

Conclusion:

Greater awareness to lab etiquette from sterile technique, tool operation, controls, to

labeling are vital to ensuring a more accurate lab. Although the lab results were inconclusive due

to the lack of data, in aiming to find our genetic lineage using PCR and gel electrophoresis, it is

clear that closer attention to lab etiquette could have allowed for more results.Without proper

sterile technique, like in the handling of the pipette tips, the samples were more prone to

contamination, which adds more genetic material for the primers to account for, potentially

causing the experiment to have another variable that cannot be accounted for. Also, without a

control, there is not a way for the results of the lab to be verified to be expected or true. Since the

control provides the reference point, especially when reading the results as +/+, +/-, or -/-, for the

gel electrophoresis, there is no way to be certain that the lab has been conducted with specific

flaws. In addition, knowing how to use your tools, like the pipette type, pipette tip, pipette

operation in regards to the stops, pipetting below the surface of the solution, or not breaking the

wells of the gel before gel electrophoresis, as well as property counterbalancing the

microcentrifuge, and closing the microcentrifuge tubes while in the centrifuge, are all some

examples of different techniques, that when paid more attention to, could have reduced

inconsistency in the results. Finally, labelling samples and recording the position of these

samples would cause less confusion and inaccuracy of testing one's own sample.