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UCR / Biochemistry / BCH 100 / What are the steps of the central dogma?

What are the steps of the central dogma?

What are the steps of the central dogma?

Description

School: University of California Riverside
Department: Biochemistry
Course: Advanced Biochemistry Laboratory
Professor: Jikui , steven song, spindler
Term: Winter 2019
Tags: biochemistry
Cost: 25
Name: BCH 162 lecture 3
Description: Experiment 1C, 1D and 2
Uploaded: 01/18/2019
48 Pages 84 Views 1 Unlocks
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LECTURE 3If you want to learn more check out Define introns.

Molecular Biology II

(Experiment 1 and 2)We also discuss several other topics like Give an example of lewis's structure.
Don't forget about the age old question of What does enso stand for?

Steps in the Mol Biol Expts We also discuss several other topics like Define elastic moduli.

OUTLINE OF THE EXPERIMENT

  • Experiment 1A: Prepare two Ampicillin-containing, LB agar plates.
  • Experiment 1B:

Generate competent E coli and transform them with plasmid DNA

  • Prepare chemically competent E. coli
  • Make the E. Coli cells competent
  • Transform plasmid DNA Into the competent E. coli
  • Plate the transformed cells and incubate the plates overnight

Plasmid preparation

  • Grow transformed cells in ampicillin supplemented medium for Day 3

  • Experiment 1C: Transformation efficiency and plasmid purification
  • Determination of transformation efficacy (count colonies), perform calculations
  • Purification of plasmid DNA using a QIAprep Spin Miniprep kit
  • Determine the concentration of the DNA
  • Experiment 1D: Polymerase Chain Reaction (PCR)
  • Experiment 2. Analyze the purity and composition of the plasmid, restriction enzyme digests and PCR products using agarose gel electrophoresis
  • Experiment 1C: plasmid purification

MaterialsDon't forget about the age old question of What is the distance between point q and line?

Thawing pellet of E. coli with pet19b (His)LDH plasmid from your starter curlture.If you want to learn more check out Who wrote an “essay on the principle of population”?

Buffers: (the content of these buffers are in the procedures below).

  1. P1
  2. P2
  3. N3
  4. PE
  5. Elution Buffer (EB)

QIAprep column is a two part column:

  1. Small inner tube containing the DNA purification resin
  2. Outer collection tube, that can be used for flow through and washes

A. The plasmid is absorbed by the resin in it

QlAprep Spin Column Plasmid Purification

1. Obtain 15 ml falcon tube containing cells from the “Experiment 1C.1'' that have been centrifugal at full speed in a table top centrifuge for 3 minutes at temperature for you by lab coordinator

2. Resuspend the pelleted bacterial cells in 250 uL buffer Pl and transfer to a

microcentrifuge tube.

Buffer Pl is: 5 mg/ml lysozyme; 10 mM EDTA: 25 mM Tris-HCL pH 8.0, 100 ug/ ml RNase A (Lysozyme hydrolyzes the 1.4-β.linkages between N-acetylmuramic acis and N-acetyl-dglucosamine in the peptidoglycan of the e. Coli cell wall, making the cells fragile: Rnase A hydrolyzes RNA.

3. Add 250 μL Buffer P2. Mix gently but thoroughly by inverting the tube 4-6 times. Do not vortex, as this will result is shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.

Buffer P2 is: 0.2 M NaOH: 1% SDS. This osmotically and chemically disrupts the cell membrane, denatures cellular protein and DNA, partially hydrolyzes RNA.

After transformation cel put in albumin media to grow, collect the culture and Resuspend (2)

(2)Purpose of PI is the lysozyme → (digest cell wall; cell fragile)

(3)P2 is very basic-disrupt cell membrane

* Binding of DNA to the resin is pH dependent at lower pH it binds strongly

4. Add 350μL Buffer N3, and mix immediately and thoroughly by inverting the tube 4-6 times.

To avoid localized precipitation, mix the solution thoroughly immediately after addition of Buffer N3. the solution should become cloudy.

Bufter N3 4.2 M Guanidine hydrochloride: 0.9 M potassium acetate: pH 4.8. This neutralizes the NaOH from the previous step, renaturing the plasmid DNA, but leaving the more complex cellular DNA denatures and insoluble and also solubilizes the protein, guanidine hydrochloride is a strong chaotrope, which encourages binding of DNA to the column resin.

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