Nov 01, 2014
Selection of Ampicillin Resistant Bacteria
External link: http://store.p212121.com/ampicillin-sodium-salt/
Protocol Citation: Sean Seaver: Selection of Ampicillin Resistant Bacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.cixufm
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: October 23, 2014
Last Modified: November 09, 2017
Protocol Integer ID: 311
Abstract
Ampicillin is a β-lactam antibiotic routinely used in bacterial selection procedures to select for bacteria (usually E. coli) that have been transformed with an ampicillin resistance plasmid (pUC19, others). Ampicillin resistance is usually due to production of beta-lactamase enzymes which cleave the beta - lactam ring rendering the antibiotic inactive.
The information below will outline preparation, storage, and a general selection procedure for ampicillin resistant bacteria.
Materials
MATERIALS
Ampicillin Sodium SaltP212121Catalog #TWA-A-301
STEP MATERIALS
Ampicillin Sodium SaltP212121Catalog #TWA-A-301
Syringe FilterP212121Catalog #LI-PES-0/22-13-S
LB Miller Agar CapsulesP212121Catalog #RP
Hot plate and stirrerP212121Catalog #BM-H4000
Petri DishP212121Catalog #LI-PD01100
Inoculating loopP212121Catalog #EM-65
Ampicillin Sodium SaltP212121Catalog #TWA-A-301
Syringe FilterP212121Catalog #LI-PES-0/22-13-S
LB Miller Agar CapsulesP212121Catalog #RP
Hot plate and stirrerP212121Catalog #BM-H4000
Petri DishP212121Catalog #LI-PD01100
Inoculating loopP212121Catalog #EM-65
Protocol materials
Inoculating loopP212121Catalog #EM-65
Inoculating loopP212121Catalog #EM-65
Syringe FilterP212121Catalog #LI-PES-0/22-13-S
Ampicillin Sodium SaltP212121Catalog #TWA-A-301
Ampicillin Sodium SaltP212121Catalog #TWA-A-301
Petri DishP212121Catalog #LI-PD01100
Hot plate and stirrerP212121Catalog #BM-H4000
Petri DishP212121Catalog #LI-PD01100
Ampicillin Sodium SaltP212121Catalog #TWA-A-301
LB Miller Agar CapsulesP212121Catalog #RP
Hot plate and stirrerP212121Catalog #BM-H4000
Syringe FilterP212121Catalog #LI-PES-0/22-13-S
LB Miller Agar CapsulesP212121Catalog #RP
Ampicillin Sodium SaltP212121Catalog #TWA-A-301
Syringe FilterP212121Catalog #LI-PES-0/22-13-S
LB Miller Agar CapsulesP212121Catalog #RP
Hot plate and stirrerP212121Catalog #BM-H4000
Petri DishP212121Catalog #LI-PD01100
Inoculating loopP212121Catalog #EM-65
An ampicillin stock solution can be prepared at a concentration of 100 mg/mL and should be stored at -20⁰C.
Add 1 g (1000 mg) of ampicillin to 10 mL of dH2O
00:03:00
Ampicillin Sodium SaltP212121Catalog #TWA-A-301
Sterilize the solution using a 0.22 μm filter
Store solution in different aliquots at -20⁰C
00:02:00
Syringe FilterP212121Catalog #LI-PES-0/22-13-S
LB-ampicillin agar preparation:
Dissolve the following in 500 mL dH2O:
5g tryptone
2.5 g yeast extract
5.0 g NaCl
7.5 g agar
25 mg ampicillin
Or
20 g pre-mixed LB agar powder (or LB agar capsules)
25 mg ampicillin
00:15:00
LB Miller Agar CapsulesP212121Catalog #RP
Boil solution on stirring hot plate for 1 – 2 min.
00:02:00
Hot plate and stirrerP212121Catalog #BM-H4000
Autoclave for 20 minutes and let cool to 50-60⁰C.
00:20:00
Pour approximately 10 mL of molten LB agar into each plate.
00:03:00
Petri DishP212121Catalog #LI-PD01100
Allow plates to solidify for approx. 20 min.
00:20:00
Selection of ampicillin resistant bacteria:
Using a sterile loop, take a sample of suspected ampicillin resistant bacteria from a colony or broth suspension and streak for isolation (using preferred method) on LB-ampicillin plates.
00:02:00
Inoculating loopP212121Catalog #EM-65
Incubate plates inverted overnight (24 hrs.) at 37⁰C.
24:00:00
Any resulting colonies should represent ampicillin resistant isolates.