PCR Lab Write-Up

Purpose:

The purpose of the lab was to gain lab experience and to learn about PCR. We

learned about PCR through using our DNA to find our alu repeats.

Hypothesis:

If I follow this procedure correctly then I will be able to see my alu repeats, which

should show I have Italian descent if I were to have it tested.

Procedure

We followed BABECs procedures on finding your own alu repeats using PCR.

To be safe in the lab, we tried to stay quiet so we could hear directions. We also made

sure we changed our pipet tip or each person so we would not have our DNA in

someone elses experiment.

Data/ Observations:
 We made a 2% agarose which we ran at 150 volt for 20 minutes. We stained it

using gel red and we ran it for 72 hours. In lanes 1A and 2A we had our ladder which

appears orange-pink in the picture. Each gel slot (1B-H and 2B-H) had a 20mL sample

of our 50mL DNA with 10 uL loading dye added to the solution. My sample was in lane

2A which had no results.

On the first day of the lab, we had to swish a salt rinse inside of our mouth to

extract cheek cells which hold DNA. The salt water tasted very bad and I dont think I

was rinsing it long enough so that could be why my lad did not go so well. I also notice

how small the Chelex beads were and how careful I had to be to make sure I did not

take any when using the pipet.

On the second day I saw the importance of keeping the PCR tube on ice

because it had to stay at 4C in order for the lab to be accurate. The tiny PCR tubes

were difficult to work with because of the size and it was hard to label it.

On the third day we made the gels to put our PCR in. The gel was opaque and it

looked similar to rosin. The spaces we had to put our PCR in was very small and it

reminded me of piping something in frosting.

Analysis:

Our class did not have enough successful results so we made theoretical data to

learn about genotype frequencies. We said there was 15 people with +/+, 10 with +/-,

and 12 with -/-. To find the frequencies you have to find how many positive or negative

alleles and divide by the total number of alleles. For the double positive and double

negative alleles, you take the number you just found and square it. For the +/- you take
the first -/- number and the first +/+ number and you multiply them together and them

multiple that be 2. The number you get is the amount of people who have that set and it

is okay if it is different from the original number. After doing those calculations with the

numbers we were given we got 11 people with +/+, 18 people with +/-, and 9 people

with -/-.

My hypothesis was not correct because I did not get any results. I feel that if I

were to try it again I would get results.

I think my one of my errors was that I did not rinse the salt rinse for a long

enough amount of time. Also I could have probably been more precise with the pipet

measurements. Next time I can also read through all the directions more closely so I

completely understand what we are doing.

We can improve this lab by making sure we are all quiet when directions are

given to us so we all hear whats going on. We can also try to manage our time better

so we are not trying to rush in the end of class. This lab could also be better if we had

more results so we can see our own results, but that can not be changed by changing

the procedure.

Conclusion:

I discovered that lab work is very precise. In this lab, we learned about PCR and

gel electrophoresis using our DNA. To do this we had to add a primer mix and a master

mix to our DNA, while heating and cooling it to exact temperatures for certain amout of

time, and b placing it into gels. One part that we had to be extremely exact on was the

time and temperatures of our PCR. If the mix was too hot or too cold, our DNA would
not react in the gels. Making sure that the temperatures and times were accurate was

crucial to the lab if you wanted to see any results. Another part of the lab where we had

to be exact was when we were using the pipet to transfer different liquids from one

place to another. The importance of that is that the lab will only work if you have the

same exact mixture of what has already been tested and proven to work. Any other

mixture will cause error because different amounts of those liquids will have diverse

reactions affecting the PCRs reaction with the gel. Now in the future I know how careful

and exact I have to be in a lab.