ELLE’S PART
Introduction:
The Amgen Biotech Experience (ABE) AP Biology Labs immerses students in different biotechnology
techniques, mirroring real-world scientific practices. These labs form a powerful sequence: Lab 1.2 B delves
into restriction enzymes, the molecular scissors of genetic engineering; Lab 4 challenges students to construct
recombinant plasmids, a fundamental skill in DNA manipulation; Lab 5 showcases bacterial transformation,
demonstrating how engineered genes can be introduced into living cells; and Lab 6 culminates in protein
purification, revealing the tangible results of genetic modifications. Together, these labs provide a
comprehensive, hands-on journey through the core principles and applications of modern biotechnology.
Hypothesis:
Lab 1.2 B: Restriction Enzyme Analysis
Purpose: How do restriction enzymes cut DNA at specific sequences?
Hypothesis: If DNA is exposed to specific restriction enzymes, it will be cut at predictable locations.
Independent Variable: Type of restriction enzyme used
Dependent Variable: Pattern of DNA fragments produced
Lab 4: Building the pARA-R Plasmid
Purpose: How can we create a recombinant plasmid?
Hypothesis: If we combine specific DNA fragments using restriction enzymes and DNA ligase, we can create a
functional recombinant plasmid.
Independent Variable: DNA fragments and enzymes used
Dependent Variable: Successful creation of the pARA-R plasmid
Lab 5: Transforming Bacteria with Recombinant Plasmids
Purpose: Can bacteria be transformed to express a new gene?
Hypothesis: If bacteria are exposed to the recombinant plasmid under specific conditions, they will take up the
plasmid and express the new gene.
Independent Variable: Presence of recombinant plasmid
Dependent Variable: Expression of the new gene in bacteria
Lab 6: Purifying the Fluorescent Protein
Purpose: How can we isolate the fluorescent protein produced by transformed bacteria?
Hypothesis: If we use chromatography techniques, we can separate and purify the fluorescent protein from
other cellular components.
Independent Variable: Purification method used
Dependent Variable: Purity and quantity of isolated fluorescent protein
Methodology:
Lab 1.2 B: Pg A-25 through A-29
Lab 4: Pg B-42 through B-45
Lab 5: Pg B-58 through B-63
Lab 6: Pg E-10 through E-14
SOLIHA’S PART
APPLE’S PART
DISCUSSION:
Lab 1.1: How to Use a Micropipette
● Summary: To introduce an important tool used in genetic engineering: the Micropipette. The
Micropipette is a tool used to transfer small, microscopic and exact volumes of liquids in either milliliters
or microliters. We used a P-20 micropipette to use the given micropipette measures for the practice
sheet.
● Hypothesis was supported
● Five tests: Pipetting five drops of different volumes (20 uL, 12.4 uL, 5.5 uL, 2.0 uL) onto a practice
sheet
● Results: Each column that represented respectively 20 uL, 12.4 uL, 5.5 uL, and 2.0 uL were perfectly
micropipetted onto the practice sheet.
● Question: Does one small difference in the numbers after the decimal point change the entire lab
process?
Lab 1.2 A/B: Gel Electrophoresis
● Summary: We were given experience with gel electrophoresis, which is a procedure in which is used to
separate and identify a mix of biomolecules (including DNA). The components of each mix are identified
by their location in the gel. We used 3 centrifuged dyes (S1, S2, S3) and pipetted them using a P-20
micropipette at 10 uL into agarose gel wells.
● Hypothesis was supported
● 3 tests: Distinguishing three centrifuged dyes (S1, S2, S3) with each other within the gel walls to see
which dye is the heaviest and the lightest
● Results: S3 was the heaviest dye, as it did not move or disperse into a lane from the well it was
pipetted into. S2 was the lightest dye as it was the dye that moved into a lane the farthest from the well
it was pipetted into.
● Question: What would happen if S3 has less solution pipetted into the well than S2?
Lab 4: Building the pARA-R Plasmid
● Summary: We labeled five clean microfuge tubes (geA-, geA+, geK-, geK+, geLIG) and added specific
respective reagents into their own tube. geK- tube has 4.0 uL dH20, 2.0 uL loading dye (LD), and 4.0 uL
K-. geK+ has the same reagents except there is 4.0 uL K+ instead of K-. geA- has 4.0 uL dH20, 2.0 uL
loading dye (LD), and 4.0 uL [Link]+ has the same reagents except there is 4.0 uL A+ instead of A-.
geLIG tube has 3.0 uL dH2O, 2.0 uL LD, and 5.0 uL ligated plasmid (LIG). All were microfuged and
dispensed at 10.0 uL of each tube into their designated walls within the agarose gel.
● Hypothesis was not supported:
● Five tests:
○ geK- tube has 4.0 uL dH20, 2.0 uL loading dye (LD), and 4.0 uL K-
○ geK+ tube has 4.0 uL dH20, 2.0 uL loading dye (LD), and 4.0 uL K+
○ geA- has 4.0 uL dH20, 2.0 uL loading dye (LD), and 4.0 uL A-
○ geA+ has 4.0 uL dH20, 2.0 uL loading dye (LD), and 4.0 uL A+
○ geLIG tube has 3.0 uL dH2O, 2.0 uL LD, and 5.0 uL ligated plasmid (LIG)
● Results: We had no ladder, as we were missing something within our testing stage.
● Question: What would happen if the non digested pKAN-R/pARA was with the tubes that were +?
Lab 5: Transforming Bacteria with Recombinant Plasmids
● Summary: Two clean microfuge tubes are labeled P- and P+. Both tubes are placed in a styrofoam cup
of ice with the CC tube. Using the P-200 micropipette, 50 uL of competent cells from the CC tube were
inputted into the P- and P+ tubes. Using the P-20 pipette, 10.0 uL of LIG was added to the P+ tube.
Both tubes were kept for 15 minutes, as three agar petri plates (LB, LB/amp, and LB/amp/ara) were set
up. LB and LB/amp plates were split into two sides (P- and P+, while LB/amp/ara plate was entirely set
up as P+. After the ice incubation of 15 minutes, the tubes are sent into a 45 second heat shock. Using
the P-200 micropipette, 150 uL of LB was added to both P- and P+ tubes. Using different tips, 50 uL of
P- and P+ respectively were added onto the respective sides of the LB plate and LB/amp plates and
spread on the respective sides. P+ was then added entirely onto the LB/amp/ara plate and spread.
● Hypothesis was supported
● 3 tests:
○ LB plate: P- and P+
○ LB/amp plate: P- and P+
○ LB/amp/ara plate P+
● Results: Only our LB/amp/ara plate had shown one single small pink colony, while the rest of our plates
were kind of empty.
● Question: Would this lab still work if the plates were heated up?
Lab 6: Purifying the Fluorescent Protein
● Summary: Spin the EC tube in the microcentrifuge for five minutes. Using the P-200 micropipette,
remove 200 uL of the supernatant from the EC tube without disturbing the cell pellet until there is no
more liquid. Add 1000 uL of E. coli to the EC tube, then microcentrifuge. Start removing more liquid
without touching the cell pellet using the micropipette. Then, using a new tip, add 150 uL of EB to the
cell pellet in the EC tube. Close the tube and drag it across the tube rack to resuspend the cells until
there are no more visible clumps. Then add 150 uL of LyB using a different tip to the EC tube, and drag
tube again. Leave overnight. Then label two clean microfuge tubes “SUPER and “RFP”. Prepare a
chromatography column. Close the valve until there is 1-2 mm of liquid left above the resin bed. Spin
EC tube for five minutes. Using the P-1000 pipette, remove 200 uL of the EC supernatant without
disturbing the cell debris pellet and put the supernatant into the SUPER tube. Using a new tip, add 200
uL of BB to the SUPER tube and mix gently. Change the volume and add 400 uL of SUPER tube mix
into the chromatography column and let the solution drain until 1-2 mm of liquid is left above the resin
bed. Using a new tip, add 1000 uL of WB into the column. Let it drain until 1-2 mm of liquid is left above
the resin bed. Then examine fluorescent protein. Using the new tip, add 1000 uL of EB twice into the
column. Let the valve drain and allow it to drain until 1-2 mm of liquid is left above the resin bed.
● Hypothesis was supported
● One test: RFP tube
● Results: Our tube was slightly pink, but not as prominently pink as the other groups, as we drained both
the column liquid and the solution we needed into the tube.
● Question: What would happen if the cell pellet was bursted at the start of the lab?
LITERATURE CITED: Amgen Biotech Experience
