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Bacterial Transformation pGLO Lab Result, Lab Reports of Genetics

Lane CollegeGenetics

DNA plasmid preps, determining DNA, concentration and purity

Typology: Lab Reports

2020/2021

Uploaded on 05/11/2021

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maya090 🇺🇸

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Lab 10: Bacterial Transformation, part 2, DNA plasmid preps,
Determining DNA Concentration and Purity
Today you analyze the results of your bacterial transformation from last week and determine
the efficiency of your transformation. You will also perform a small-scale DNA plasmid preparation,
which involves isolating a DNA plasmid from bacterial cells (effectively the opposite of
transformation). Finally, you will use the spectrophotometer to determine the concentration and
purity of a DNA sample.
Activity 10a
Transformation, Part 2: Calculating Transformation Efficiency
Purpose
The purpose of this activity is to observe the results from the bacterial transformation you
performed last week. You will observe the four agar plates to see if the results match the
predictions you made last week. You will also calculate the efficiency of your transformation to
quantitatively determine the relative success of the transformation procedure.
Procedure
1. Use a data table similar to Table 10.1 below to record data from your bacterial transformation
experiment. Observe your group’s plates using the UV lamp and record what you see on each of
the four plates. In your observations, note whether you see bacterial colonies, and whether the
colonies fluoresce (glow) green under the UV light.
Table 10.1 Bacterial Transformation Observations
Plates
Observations
+pGLO, LB/amp
+pGLO, LB/amp/ara
-pGLO, LB/amp
-pGLO, LB
pf3
pf4
pf5

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Lab 10: Bacterial Transformation, part 2, DNA plasmid preps,

Determining DNA Concentration and Purity

Today you analyze the results of your bacterial transformation from last week and determine the efficiency of your transformation. You will also perform a small-scale DNA plasmid preparation, which involves isolating a DNA plasmid from bacterial cells (effectively the opposite of transformation). Finally, you will use the spectrophotometer to determine the concentration and purity of a DNA sample.

Activity 10a

Transformation, Part 2: Calculating Transformation Efficiency

Purpose The purpose of this activity is to observe the results from the bacterial transformation you performed last week. You will observe the four agar plates to see if the results match the predictions you made last week. You will also calculate the efficiency of your transformation to quantitatively determine the relative success of the transformation procedure. Procedure

  1. Use a data table similar to Table 10.1 below to record data from your bacterial transformation experiment. Observe your group’s plates using the UV lamp and record what you see on each of the four plates. In your observations, note whether you see bacterial colonies, and whether the colonies fluoresce (glow) green under the UV light. Table 10.1 Bacterial Transformation Observations Plates Observations +pGLO, LB/amp +pGLO, LB/amp/ara
  • pGLO, LB/amp
  • pGLO, LB
  1. Calculate your transformation efficiency. There are several steps involved in calculating transformation efficiency. Step 1: Determine the total number of green fluorescent cells. Place your LB/amp/ara plate near the UV light source. Each colony on the plate can be assumed to be derived from a single cell. That reproduced many times to form a colony. Therefore, counting the number of green fluorescent colonies tells you how many cells were transformed. Step 2: Determine the total amount of pGLO plasmid DNA added to the bacterial cells on the LB/amp/ara plate. a) In our transformation, we used 10 μL of pGLO DNA solution (approximate amount in the loopful in step 5) at a concentration of 0.08 μg/μl. Calculate the total μg of DNA that we used in the experiment. Total amt. of DNA (μg) = (concentration of DNA (μg/μl) x volume of DNA in μl) = b) Determine the fraction of this total amount of DNA that was actually spread onto the LB/amp/ara plate. On your LB/amp/ara plate, you spread 100 μl of (+pGLO) transformation suspension from a total volume of ~510 μl. Fraction of DNA spread on plate = Volume spread on LB/amp/ara plate Total volume in transformation tube = c) Determine the number of micrograms (μg) you spread on the LB/amp/ara plate. To do this, multiply the total amount of DNA used in the experiment (from part a above) by the fraction of DNA you spread on the LB/amp/ara plate (from part b above). pGLO DNA spread (μg) = Total amt of DNA used (μg) x fraction of DNA on plate = d) Calculate transformation efficiency. Transformation = total # of cells growing on LB/amp/ara plate = ________________ Efficiency amount of DNA spread on LB/amp/ara plate = transformants/μg DNA

Total # of cells transformed

(colonies counted) =

  1. Centrifuge for 10 minutes at high speed. During the centrifugation, place a spin column in a 2 mL collection tube.
  2. Bind. Apply the supernatant from step 6 to the spin column by micropipetting. In this step, the plasmid DNA binds to the column; other molecules should not bind and “flow through.”
  3. Centrifuge 30-60 seconds. Discard the flow-through.
  4. Wash column by adding 500 μl of Buffer PB. Centrifuge 30-60 seconds. Discard the flow- through.
  5. Wash column again with 750 μL of Buffer PE. Centrifuge 30-60 seconds. Discard the flow- through.
  6. Centrifuge an additional 1 minute to remove residual wash buffer.
  7. Elute. In this step, the DNA is released from the column by adding a low-salt or no-salt liquid. Place spin column into a clean 1.5 ml microcentrifuge tube. Add 50 μL of Buffer EB ( mM Tris, pH 8) to the center of the column. Let stand for 1 minute, and centrifuge for 1 minute.

Activity 10c

Determining DNA Concentration and Purity

Purpose In this activity, you will learn how to calculate the concentration of a DNA sample using the spectrophotometer. You will also use the spectrophotometer to determine the degree of purity of a DNA sample. Background Whenever technicians purify DNA samples, they then need to determine: 1) how much DNA they obtained, and 2) how pure the DNA is (meaning, are there contaminating molecules, usually protein or RNA, present?). This can be done using a UV spectrophotometer (one that can measure absorbance at light wavelengths in the UV range. Procedure

  1. Determination of DNA concentration To measure the concentration of DNA in solution, the absorbance is measured (in absorbance units, or au) at 260 nm. Since 50 μg/ml of DNA in solution gives an absorbance of 1 au, the following equation can be used to give the DNA concentration in μg/ml: DNA concentration 50 μg/mL = X μg/mL equation 1 au au at 260 nm EX: the absorbance of a sample of DNA solution at 260 nm is measured as 0.25 au. What is the concentration of DNA in the sample? 50 μg/mL = X μg/mL = 0.25 x 50 μg/mL = 12.5 μg/mL 1 au 0.25 au
  2. Determination of DNA purity The test to determine the purity of a DNA sample is a simple calculation of the ratio of absorbance at 260 nm (the wavelength of DNA absorbance) to the absorbance at 280 nm (the wavelength of protein absorbance). This is called a 260/280 reading. DNA purity abs at 260nm equation abs at 280 nm If the 260/280 ratio is approximately 1.8, the DNA sample is considered fairly pure. If it’s too high, RNA contamination is suspected. If it’s too low, protein contamination is suspected. Your instructor will give you additional instructions during lab and will help you measure the absorbance of your sample at 260 nm and 280 nm on the UV spectrophotometer. After you have recorded the data in your notebook, calculate your DNA concentration and DNA purity and compare with other groups to see how effective your miniprep procedure was at purifying the DNA from the transformed bacterial cells.
  1. ( continued ) Given this data, calculate the transformation efficiency.
  2. Looking over the steps of the miniprep protocol, describe what is happening in each of the following steps: a) Lysis b) Neutralization c) Binding d) Elution
  3. After purifying plasmid DNA from bacterial cells, you measure the absorbance of the DNA solution in a UV spectrophotometer at 260 nm and at 280 nm. The absorbance at 260 nm measures 1.5. The absorbance at 280 nm measures 1.0. a) What is the concentration of the DNA? b) Comment on the purity of the DNA.