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Latest revision as of 06:58, 19 October 2016

Transformation Efficiency · Benchling

Transformation Efficiency

Introduction

Transformation efficiency is the efficiency by which bacterial cells take up foreign DNA and express the genes encoded by it. Since in reality only a small portion of competent cells are transformed, a reasonable representation of transformation efficiency is calculated by dividing successful transformants by the amount of DNA used during transformation procedure, in CFU/μg of DNA. By verifying the transformation efficiency value against that of standard competent E.coli cells (1.5x10^8 to 6x10^8 cfu/μg DNA), competency of electroporated sample can be validated. The transformation efficiency kit from iGEM includes five vials of purified DNA from BBa_J04450 (RFP construct) in plasmid backbone pSB1C3. Each vial contains DNA at a different concentration: 50pg/ul, 20pg/ul, 10pg/ul, 5pg/ul, 0.5pg/ul. Transformation efficiency test is performed with each concentration of DNA in parallel. Safety Precausions SDS (Safety data sheet): Refer to the SDS sheets for all listed materials before entering the lab. Be prepared to answer any questions regarding the information on these sheets. PPE (Personal protective equipment): Proper lab attire should be worn throughout the experiment: This means that upon entering the lab you should be wearing long pants and close-toed shoes. Contact lenses should not be worn. Furthermore, a lab coat, goggles, and gloves should be worn at all times, and long hair should be tied back. Disinfect lab bench with 70% ethanol prior to use. Hazards:

Materials

  • Reagents
    • 70% ethanol
    • Ice
    • Competent cell alequot(s)
    • SOC media
  • Equipment
    • Paper towels
    • 5x 2.0ml microcentrifuge tube
    • Transformation Efficiency Kit
    • Agar plates with chloramphenicol
    • Waterbath with thermometer
    • Incubator
    • spreader
    • Pipettor P10, P100, P1000 and pipette tips
    • Lab marker
    • Ice bucket

Procedure

  • Protocol
  1. Spin down the DNA tubes from the Transformation Efficiency Kit to collect all of the DNA into the bottom of each tube prior to use. A quick spin of 20-30 seconds at 8,000-10,000 rpm will be sufficient. Note: There should be 50 μL of DNA in each tube sent in the Kit.
  1. Thaw competent cells on ice. Label one 2.0ml microcentrifuge tube for each concentration and then pre-chill by placing the tubes on ice.
  1. Pipet 1 μL of DNA into each microcentrifuge tube. There will be 5 microcentrifuges that contain 1 μL of DNA of different concentration.
  1. Pipet 50 μL of competent cells into each tube. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath so that thermometer reads 42°C.
  1. Heat-shock the cells by placing into the waterbath for 1 minute. Be careful to keep the lids of the tubes above the water level, and keep the ice close by.
  1. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover.
  1. Add 200 μL of SOC media per tube, and incubate at 37°C for 2 hours. Prepare the agar plates during this time, also label them with concentration of DNA used.
  1. Pipet 20 μL from each tube onto the appropriate plate, and spread the mixture evenly across the plate with spreader. Incubate at 37°C overnight or approximately 16 hours. Position the plates so the agar side is facing up, and the lid is facing down.
  1. Count the number of colonies on a light field or a dark background, such as a lab bench. Use the following equation to calculate your competent cell efficiency.  Transformation efficiency = (colonies on one plate) / ng of DNA plated x 1000ng/μg  where ng of DNA plated = 1 μL x concentration of DNA (refer to vial) x (volume plated (20 μL)/ total reaction volume(251 μL))  Note: The measurement "ng of DNA plated" refers to how much DNA was plated onto each agar plate, not the total amount of DNA used per transformation.
  • Evaluation
  • Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/μg DNA, where "cfu" means "colony-forming unit" and is a measurement of cells. Here are some good sample results:
A
B
C
D
E
F
1
DNA concentration0.5pg/μL5pg/μL10pg/μL20pg/μL50pg/μL
2
# od colonies10-20120-170280-360480-802500-1000+
Table1
  • Leaving the lab
  • Prior to leaving the lab, you should:
  • 1. Clean dirty glassware, or at least set aside the glassware to be cleaned by a designated individual.
  • 2. Wipe down your workspace.
  • 3. Ensure that all materials have been returned to their places, and that the plates have been properly stored in the fridge.
  • Acknowledgement
  • Made by Mark Wang (IGEM 2015)
  • Protocol taken with minor modification from iGEM guideline
  • Changelog
  • Created 5/17/2016



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