Haynes Lab:Notebook/Shawn Cloning bootcamp

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Project Description/Abstract

  • Cloning Bootcamp at Dr.Haynes lab with Ben Nyer.
  • The objective of stage A is to produce a large batch of replicated DNA and confirm that the batch is undamaged.
  • The method to compete this task is to change bacteria by allowing it to undergo the process of transformation with the plasmid that codes for a specific antibiotic resistance. Expose the bacteria to the antibiotic to single out the transformed bacteria and allow the cells to grow in LB, or liquid medium. Extract the DNA from the cell bodies using a miniprep kit. Finally analyze and document the parts for the recreated DNA.

Notes


====Data 1====
BioBrick: KAH126/mv2
Vector Backbone: MV2
Antibiotic resistance gene: Ampicillin

Samples transformed:
Sample One = Plasmid
Sample Two = Negative control


====Data 2====

Results of:
Sample One = Colonies
Sample Two = No growth

Liquid Cultures:
Culture One = From colony one, KAH126/mv2, Ampicillin
Culture Two = From colony two, KAH126/mv2, Ampicillin


====Data 3====



DNA Concentration Data

Plasmid OD260 OD260/280 ng/μL
1. Plasmid 1 .4355 1.901 435.2575
2. Plasmid 2 .186 1.9245 186.0375

OD260/280 measures the purity if the sample which should be around 2.
ng/μL measures how much product was yielded.


Restriction Digest Table

  • Checked plasmid minipreps with EcoRI/PstI digests
Reagent Volume Expected:
1. Plasmid 1 = 1500
2. Plasmid 2 = 4500
Today's gel
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 3.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 8.5
  15 μL --> 37°C/ 15 min.


DNA Sequencing Samples

  • Submitted to DNASU on: 'date'
  • Order Number:
  1. Plasmid 1 - forward primer 'name'
  2. Plasmid 1 - reverse primer 'name'
  3. Plasmid 2 - forward primer 'name'
  4. Plasmid 2 - reverse primer 'name'




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