practicum 2

Question 1

Solution I (Resuspension buffer)

Answer 1

Contains glucose (maintains osmotic balance), Tris (pH buffer), and EDTA (chelates Mg²⁺ to inhibit DNases). It gently resuspends cells and prepares them for lysis.

Question 2

Solution II (Lysis buffer)

Answer 2

Contains NaOH (alkaline) and SDS (detergent). It lyses the cells and denatures DNA and proteins.

Question 3

Solution III (Neutralization buffer)

Answer 3

Contains potassium acetate. Neutralizes the mixture, causing chromosomal DNA, proteins, and cell debris to precipitate. Plasmid DNA remains in solution.

Question 4

Other Components: Ethanol, Express Matrix glass beads

Answer 4

• Ethanol: Used to precipitate DNA from the aqueous solution after the debris is removed.
  -Express Matrix glass beads: Help bind and purify plasmid DNA during miniprep.

Question 5

When are components removed?

Answer 5

• Bacterial DNA and cellular debris: Removed after neutralization and centrifugation (Solution III).
• Bacterial RNA: Removed by RNase or washed away during purification (often in early steps or wash buffers).

Question 6

Restriction Enzymes

Answer 6

• Proteins that cut DNA at specific sequences (recognition sites). - - They’re used to digest plasmid DNA to analyze or manipulate it.
• Enzymes used in this lab (typically):
  Examples could be EcoRI, HindIII, BamHI????

Question 7

Gel Electrophoresis Purpose:

Answer 7

To separate DNA fragments based on size (length in base pairs).

Question 8

Agarose function

Answer 8

the gel matrix; DNA migrates through its pores.

Question 9

TBE buffer

Answer 9

Maintains pH and ionic strength; conducts current

Question 10

Loading dye

Answer 10

dds color and density so the sample sinks and is visible during loading

Question 11

DNA ladder (500 bp standard)???

Answer 11

A set of known fragment sizes used to estimate unknown DNA sizes.

Question 12

GelRed

Answer 12

DNA stain that fluoresces under UV light to visualize bands.

Question 13

Electrophoresis Setup

Answer 13

• Horizontal gel is used for DNA (as opposed to vertical for proteins).
• Band = Many identical DNA fragments of the same size.
• Apply current: DNA (negatively charged) moves toward the positive (red) electrode.
• Smaller fragments migrate faster (further), larger ones slower (less distance).

Question 14

UV Light Box

Answer 14

Used to visualize GelRed-stained DNA bands, as they fluoresce under UV.

Question 15

Purpose of This Lab

Answer 15

To isolate plasmid DNA (miniprep), verify its identity via restriction digestion, and analyze it using gel electrophoresis.

Question 16

Express Matrix

Answer 16

Used to selectively bind plasmid DNA during purification for easier isolation.

Question 17

Restriction Endonucleases

Answer 17

• Enzymes that recognize specific DNA sequences and cleave both strands.
• You likely used enzymes such as EcoRI, HindIII, and BamHI.

Question 18

How Gel Electrophoresis Works

Answer 18

• DNA migration: From negative (black) to positive (red) end.
• DNA is pulled through agarose based on size — smaller = faster/farther.
• GelRed allows bands to be visualized under UV light.

Question 19

Why Add These Reagents?

Answer 19

-GelRed: Binds DNA for visualization.
-Loading dye: Helps with tracking and ensures sample sinks.
-DNA standards: Used to create a standard curve and estimate unknown sizes.

Question 20

Standard Curve Explanation

Answer 20

• Measure the distance each DNA ladder band migrated (in mm).
• Plot log₁₀ of base pair size vs. distance migrated on semilog graph paper.
• Fit a line to the standard curve.
• Use it to determine: The size of unknown DNA fragments by measuring their migration and comparing to the curve.

Question 21

Interpreting a Gel

Answer 21

• More bands = multiple cuts by enzyme.
• One band = no cut (supercoiled or uncut plasmid).
• Band intensity: Thicker = more DNA.
• Migration speed: Shorter DNA travels farther.