Sep 17, 2015
Transform Stratagene's XL-10 Gold Ultracompetent cells
- Harold Bien1
- 1Stony Brook University
Protocol Citation: Harold Bien 2015. Transform Stratagene's XL-10 Gold Ultracompetent cells. protocols.io https://dx.doi.org/10.17504/protocols.io.dtz6p5
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: September 16, 2015
Last Modified: March 28, 2018
Protocol Integer ID: 1625
Abstract
Protocol for transforming XL-10 GOld Ultracompetent cells from Stratagene (now Agilent). Protocol adopted from manufacturer's instructions.
Guidelines
Use of 14-ml BD Falcon polypropylene round-bottom tubes: It is important that 14-ml BD Falcon polypropylene round-bottom tubes (BD Biosciences Catalog #352059) are used for the transformation protocol, since other tubes may be degraded by β-mercaptoethanol. In addition, the duration of the heat pulse has been optimized using these tubes.
Aliquoting Cells: Keep the cells on ice at all times during aliquoting. It is essential that the polypropylene tubes are placed on ice before the cells are thawed and that the cells are aliquoted directly into pre-chilled tubes. It is also important to use 100 μl of cells per transformation. Decreasing the volume will reduce efficiency.
Use of β-Mercaptoethanol (β-ME): β-ME has been shown to increase transformation efficiency. The β-ME mixture provided is diluted and ready to use. Stratagene cannot guarantee results with β-ME from other sources.
Use of NZY+ Broth: Transformation of the supplied ultracompetent cells has been optimized using NZY+ as the medium for outgrowth following the heat pulse. Substitution with another outgrowth medium may result in a loss of efficiency.
Quantity and Volume of DNA: The greatest efficiency is obtained from the transformation of 1 μl of 0.01 ng/μl supercoiled pUC18 DNA per 100 μl of cells. When transforming a ligation mixture, add 2 μl of the ligation mixtureper 100 μl of cells. A greater number of colonies may be obtained by transforming up to 50 ng DNA, although the resulting efficiency (cfu/μg) may be lower. The volume of the DNA solution added to the reaction may be increased to up to 10% of the reaction volume, but the transformation efficiency may be reduced.
Heat Pulse Duration and Temperature: Optimal transformation efficiency is observed when cells are heat-pulsed at 42°C for 30 seconds. Efficiency decreases sharply when cells are heat-pulsed for <30 seconds or for >40 seconds. Do not exceed 42°C.
Plating the Transformation Mixture: If plating <100 μl of cells, pipet the cells into a 200 μl pool of medium and then spread the mixture with a sterile spreader. If plating â¥100 μl, the cells can be spread on the plates directly. Tilt and tap the spreader to remove the last drop of cells. If desired, cells may be concentrated prior to plating by centrifugation at1000 rpm for 10 minutes followed by resuspension in 200 μl of NZY+ medium.
Materials
STEP MATERIALS
NZY broth
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314
XL-10 Gold Beta-mercaptoethanol mixAgilent Technologies
NZY broth
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314
XL-10 Gold Beta-mercaptoethanol mixAgilent Technologies
Protocol materials
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314
XL-10 Gold Beta-mercaptoethanol mixAgilent Technologies
NZY broth
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314
XL-10 Gold Beta-mercaptoethanol mixAgilent Technologies
NZY broth
NZY broth
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314
XL-10 Gold Beta-mercaptoethanol mixAgilent Technologies
Pre-chill 14mL sterile culture tubes on ice
Pre-heat 0.9mL of NZY+ broth to 42°C
1 mL
NZY broth
Thaw XL-10 Gold Ultracompetent cells on ice then mix gently after completely thawed
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314
Aliquot cells into pre-chilled sterile culture tubes
100 µL
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314
Add beta-mercaptoethanol
4 µL
XL-10 Gold Beta-mercaptoethanol mixAgilent Technologies
Swirl tube gently then incubate on ice for 10 minutes swirling gently every 2 minutes
00:10:00
Add 0.1-50ng of DNA or 2uL of ligation product
2 µL
Swirl gently then incubate on ice for 30 minutes
00:30:00
Heat pulse tube at 42°C for exactly 30 seconds. The duration of the heat pulse is critical.
00:00:30
Incubate cells on ice for 2 minutes
00:02:00
Add 0.9mL of pre-heated NZY+ broth to each tube
1 mL
Incubate at 37°C for 1 hour with shaking at 225-250 rpm
01:00:00
Plate no more than 200uL of transformation mixture per LB-agar plate with antibiotic selection
Incubate plates at 37°C overnight.
15:00:00