Wolbachia Lab Report by voldemortwand007


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									Ashwin Suresh                           18 December 2012                                  Period 7
                                   Wolbachia Lab Report

       The purpose of this lab

         Wolbachia is a parasitic bacterium that infects 20% of known insect species. Wolbachia
interferes with a variety of tissues but mainly the sex organs. An infected sperm cell cannot
properly produce offspring with an uninfected egg cell. However, when an infected sperm cell
and an infected egg cell merge, they result in perfectly fine offspring. Therefore, infected
females produce more offspring. In many insect species, unfertilized eggs result in males and
fertilized eggs result in females. The unfertilized eggs of an infected female always duplicate
their chromosomes always resulting in female offspring. The primary effect of Wolbachia is
insect populations with a high percentage of females. (Timmer Arstechnica.com)
         The shape of a DNA molecule is a double helix consisting of nucleotides. Each
nucleotide has a sugar, deoxyribose, and a phosphate group which make up the backbone of the
DNA molecule. Each nucleotide also has a nitrogenous base, which could be adenine, guanine,
cytosine, or thymine. The nitrogenous bases are key to how the DNA molecule stores
information. Each DNA molecule is arranged so that adenine (A) is opposite thymine (T) and
guanine (G) is opposite cytosine (C). During the replication (S) phase of the cell cycle the
enzyme helicase separates the opposite nucleotides resulting in two single helix strands. Each
side is used as guide for a new complementary strand to be attached to it. DNA polymerase
matches up corresponding nitrogenous bases; A to T and G to C. DNA ligase joins the
nucleotides into one strand opposite of the original. A similar process is used when the cell reads
the DNA. RNA polymerase separates the DNA strands and uses one strand as a template to
create mRNA. mRNA diffuses into the cytoplasm where it is read by transfer RNA. Each three
base sequence on the mRNA, called a codon, code for a different amino acid. The amino acids
are then assembled to form a protein. The structure of DNA allows it form many different
proteins, thus creating very complex organisms.
         In a eukaryotic cell, the double helix is wound around proteins called histones and then
further compiled into nucleosomes, then coils, then supercoils, and finally chromosomes which
are visible during cell division. Prokaryotic DNA is not wrapped around any histones therefore it
is not formed into chromosomes. Eukaryotic DNA is linear and is contained in a number of
chromosomes. Prokaryotic consists of only a single circular DNA molecule and a variety of
smaller plasmids. DNA in a eukaryotic cell is enclosed within a nucleus, while prokaryotic cells
do not have a nucleus. (Fancher)
         PCR (Polymerase Chain Reaction) is a process that amplifies a segment of DNA into
billions of identical copies. This enables scientists to study certain genes in an entire genome.
The materials involved in PCR are nucleotides, primers, and Taq polymerase. Primers are
usually 20 nucleotides long and are complementary to the ends of the DNA that is wished to be
amplified. A PCR reaction takes up several hours and has twenty to thirty-five cycles. The first
step of a cycle is to heat the DNA to 95o Celsius, separating the hydrogen bonds between the two
strands. The second step is too cool down the mixture to 60oC, allowing the primers to bond to
their complementary sequence on each DNA strand. The third step consists of raising the
temperature to 72oC so the Taq polymerase can add nucleotides to the 3’ end of each strand.
After four cycles, half the DNA strands contain only the target DNA. The number of target DNA
strands increases exponentially after more cycles.
Ashwin Suresh                           18 December 2012                                   Period 7
         During gel electrophoresis, one side of the gel has positive charge and one side of the gel
has a negative charge. DNA that has reacted with restriction enzymes is placed at the negative
end of the gel in wells. The electric current is then turned on. Because DNA has a negative
charge from the phosphate group, it is repelled from the negative side and attracted to the
positive side. The restriction enzymes cut up the DNA where ever it reads the sequence
“GAATTC”. The shorter pieces of the DNA move faster through the porous gel and end up
closer to the positive side. The longer strands take longer to navigate through the gel and end up
closer to the negative side. DNA from different individuals have this sequence at different points
in their DNA. Therefore, each individual has a unique pattern of DNA segment sizes resulting in
a unique pattern of bands on the gel.

    If the sample arthropods are infected with Wolbachia, then we expect to find additional
       bands on the gel other than the bands for mitochondrial DNA.
    If the sample arthropods are not infected with Wolbachia, then we only expect to see the
       bands from the mitochondrial DNA.

Materials and Method______________________________________________________
        The first part of the lab is DNA extraction. Get two microcentrifuge tubes and number
them 1 or 2 in addition to your period and group number. Use P200 pippettor to pipet 180
microliters of buffer ATL into each tube. ATL is a detergent that breaks down the insect. If the
insect was stored in ethanol, blot away the ethanol with a Kimwipe. Do the same for the + or –
Nasonia. Using tweezers place the sample insect into tube 1 and the nasonia into tube 2. Using a
microtube pestle, macerate the insect in tube 1. It is important to do this step quickly, because
macerating the insect frees DNases that eat DNA. Immediately after the previous step, add 20
microliters of proteinase K. Proteinase K is an enzyme that breaks down histones to unravel the
DNA. Proteinase K also breaks down DNases to prevent the DNA from being harmed. Now, add
200 microliters of buffer AL to the mixture. Buffer AL is a detergent that breaks down the
phospholipid membranes, which are made of fatty acids. Vortex the mixture for ten seconds.
Repeat for the nasonia in tube 2. Incubate both tubes at 70oC for at least ten minutes. After the
tubes are done in the incubator, add 200 microliters of 95% ethanol to each tube. Ethanol is polar
and extracts the DNA from the other content in the tube. Vortex for ten seconds. Store the tubes
at 4oC overnight. Get two spin columns fitted in 2.0 milliliter collection tubes. Using a Sharpie
marker, label the lid of each spin column 1 or 2 along with your period and group number. Using
a P1000 pippetor, pipet 600 microliters of liquid from yesterday’s “1” tube into spin column 1.
Centrifuge the spin column at 8,000 rpm for one minute. The DNA is now stuck to the filter of
the spin column. Take out the tube and dispose of the waste in the collection tube. Place the spin
column back into the same collection tube that you just emptied. Pipet 500 microliters of buffer
AW1 into the spin column. AW1 denatures the proteins still mixed with the DNA. Centrifuge for
one minute at 8,000 rpm. The DNA is still caught on the filter of the spin column. Take out the
tube and empty the waste from the collection tube. Place the spin column back into the same
collection tube that you just emptied. Pipet 500 microliters of buffer AW2 into the spin column.
AW2 is another wash buffer that helps purify the DNA. Centrifuge for three minutes at 14,000
rpm. Take out the spin column and place them into a labeled 1.5 milliliter microcentrifuge tube.
Pipet 100 microliters of buffer AE into the spin column. The AE is an elution buffer and unsticks
the DNA from the filter and into the microcentrifuge tube. Incubate the tube at room temperature
for one minute. Centrifuge at 8,000 rpm for one minute. Discard the spin column but keep the
labeled microcentrifuge tube that contains the DNA. Store at -20oC until ready for PCR. Pipet
Ashwin Suresh                           18 December 2012                                  Period 7
600 microliters of liquid from yesterday’s “2” tube into spin column 2. Repeat the same steps for
spin column 1.
        The second part of the lab is the polymerase chain reaction or PCR. Get two PCR ready
tubes and label like all the other tubes. Each PCR tube contains a pellet, that is preformulated
with Taq polymerase, MgCl2, Buffer, and dNTPs. Pipet 23 microliters of master mix into each
PCR tube. The master mix contains 12 microliters (5 micrometers) of the primers, Wspec-F,
Wspec-G, CO1-F, CO1-R. The master mix also contains 90 microliters of sterile water. The
Wspec primers correspond to a DNA sequence unique to Wolbachia. The CO1 primers
correspond to a region in mitochondrial arthropod DNA that codes for the protein, cytochrome
oxidase I. Pipet, using a P20 pippettor, 2 microliters of DNA from microfuge tube 1 into PCR
tube 1. Do the same for the nasonia DNA in microfuge tube 2. Close and tap the tubes gently to
mix the components in the tube. Place the PCR tubes in the thermal cycler. The first cycle should
be of temperature 94oC and last two minutes. The heat is strong enough to break the hydrogen
bonds between the nucleotides, thus separating the strands. The second cycle should last two
minutes and fifteen seconds. The first thirty seconds should be 94oC again to break the hydrogen
bonds between the nucleotides, thus separating the strands. The next 45 seconds should be at
55oC. This lower temperature allows for the primers to attach to the DNA strands. The next
minute should be at 72oC, which is the best temperature for Taq polymerase to attach nucleotides
to the existing strand. Each cycle of your next 29 cycles should look like this. The last (32nd)
cycle should last ten minutes at 72oC. Finally after the thermal cycler process is completed, about
two hours, store the PCR tubes at -20oC. The Polymerase chain Reaction is now over.
        The third and final part of the lab is gel electrophoresis. First, pipet 2 microliters of

       All the results from our Period 7 class failed.
          Ashwin Suresh                                     18 December 2012                                   Period 7

Class &                                                                                  Arthropod                Results    Results
                                        Prepared by                                                    notes                  (+/-)
Group                                                                                      Order                 (+/-) COI   Wolbachia
            Thomas Yan, Ricky Yang, Jan Clasen, Theo DeFuria, Luke
  1-1                                                                                    arachnida                   -           -
                                Zheng, Erik Viggh
          Michelle Yao, Rose Zhao, Shawnae Ma, Brianna Hoff and Judie
  1-2                                                                                     diptera                    -           -
              Mark Benati, Tharun Sankar, Ben Fanucci-Kiss, Robert
  1-3                                                                                   hymenoptera                  -           -
                  Tabachnik, Siddharth Limaye, and Jeffrey Shao
  1-4                                                                                   lepidoptera                  -           -
            Madhuri Jois, Michelle Wu, Pelin Ozturk, Jennifer Ma, Kaitlin
  1-5                                                                                   lepidoptera                  -           -
           Talia Quaadgras, Hannah Hoffman, Sarah Mamlet, Stephanie
  1-6                                                                                   hymenoptera                  -           -
                          Margolis, and Brooke Blackshaw
  2-1                                                                                   hymenoptera                  -           -
  2-2                                                                                   hymenoptera                  -           -
          Emily Liu, Alli Sedler, Jack Hoggard, Matt Bullock, Sam Toulmin,
  2-3                                                                                    orthoptera                  -           -
                                      Josh Linker
  2-4                                                                                   hymenoptera                  -           -
  2-5                                                                                      isopoda                   -           -
  2-6                                                                                    lepidoptera                 -           -
            Jack Billings, Marshall Grant, Gabe Kline, Nick Duncan, and
  3-1                                                                                     isopoda                    -           -
                                     Melody Mao
  3-2            Grace Wu, Julie Yao, Carol Zhang, Fiona Koshy                             diptera                   -           -
  3-3                                                                                   hymenoptera                  -           -
  3-4                                                                                    orthoptera                  -           -
  3-5                                                                                   hymenoptera                  -           -
            Teja Pallikonda, Nina Prakash, Roshni Sahoo, and Maggie
  3-6                                                                                     isopoda                    -           -
  4-1                                                                                    coleoptera                  -           -
  4-2          Akshay Karthik, William Hu, Ajay Suresh, Alex Mateev                     hymenoptera                  -           -
  4-3                                                                                    orthoptera                  -           -
          Michelle Chen, Rebecca Kane, Avani Khatri, Vanessa Low, Julia
  4-4                                                                                   hymenoptera                  -           -
  4-5                                                                                    hemiptera                   -           -
  4-6                                                                                   hymenoptera                  -           -
                Emily Ropiak, Selena Gonzales, Charles Watt, Ellen
  7-1                                                                                     Isopoda                    -           -
                            McCormick,Bekah Pierce
  7-2                                                                                   Heteroptera                  -           -
            Kevin Hu, Akash Purohit, Shirley Xu, Ezra Stein, and Sam
  7-3                                                                                     Isopoda                    -           -
           Srinivas Setty, Saahil Claypool, Dylan Caldwell, David Chen,
  7-4                                                                                   Hymenoptera                  -           -
                           Darren Yang, Ashwin Suresh
  7-5                                                                                    Arachnid                    -           -
                Jamie Young, Julian Waugh, Abishek Kumar, Varan
  7-6                                                                                   Lepidoptera                  -           -
                 Culanathan, Kevin Leung , and Bobby Smieszny
  8-1      Ankit Datta, Matt Gilligan, Richard Tang, Valerie Han, Sarah Gao, Anuradha   Hymenoptera                  -          -
  8-2                                                                                   Lepidoptera                  -          +
              Julianne Higgins, Tara Jawahar, Katya Muzin, Vennela
  8-3        Pandaraboyina, Peter Rakauskas, Leanne Quinn and Ben                        Coleoptera                  -           -
  8-4                                                                                     Diptera                    -           -
      Ashwin Suresh                           18 December 2012                   Period 7
8-5                                                                    Diptera        -     -
8-6   Artem Petrov, James McClung, Alex Kim, Sri Nevvula, Joie Liba   Isopoda         -     -
     Ashwin Suresh                  18 December 2012                        Period 7

     Conclusion____________                                                 _____

Literature Cited__________________________________________________________________
Ashwin Suresh                          18 December 2012                                   Period 7

Here are a few ways enzymes play a role in the human body. You have probably heard about
people that are lactose intolerant. These people cannot consume dairy products because their
body cannot digest it. In order for the body to digest it, the disaccharide lactose must be broken
down into the monosaccharaides glucose and galactose. This is done with the help of the enzyme
lactase, something that lactose intolerant people lack. This shows how important enzymes are
because not having lactase prevents you from eating a whole entire category of foods.

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