User:Jarle Pahr/Transformation

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Notes on bacterial transformation:

1 h incubation has been mentioned to be sufficient for recovery of Kan resistant transformants:

See also

Kanamycin works by interfering with protein translation.

High efficiency transformation of Escherichia coli with plasmids:

Comparison of protocols:

Protocol source Competent cells DNA amount Pre-heatshock incubation Heat shock Post heat-shock incubation Reference
iGEM 50 uL 1-2 uL (resuspended DNA from iGEM kit) 30 minutes. 42 C for 60s. On ice for 5 min. Add 200 uL SOC, 37 C for 2 h.
AddGene 20-50 uL 1-5 uL (10 pg to 100 ng) 20-30 min 42 C 30-60 s On ice for 2 min. Add 200-250 uL SOC, 37 C for 45 min.

Commercial competent cells

Name Description/Use Supplier (Main) Genotypes Transformation efficiency (cfu/ug) Comment Reference
XL10-GOLD Ultracompetent cells Chemocompetent cells Stratagene Hte >5x10^9

Preparation of competent cells

Snap freezing may help avoid formation of ice crystals?

Inoue 1990.:

Chung et al.:

Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors.:


Significant variables in transformation:

  • Incubation time pre-heat shock
  • Heat shock
  • Incubation time and conditions post-heat shock.

Effect of post-heat shock incubation: Ampicillin-resistant transformants can be plated directly, as ampicillin only affects growing cells. Other selection markers, such as Kanamycin, may require a post-heatshock incubation to allow outgrowth and establishment of resistance.

Factors affecting choice of incubation time:

If several plasmids (re-ligation and desired construct) are present in the ligation mix, the proportion of the major species will increase with longer incubation times, assuming exponential growth.

To increased consistency and easier quality control, consider using larger aliquots (500 uL) supercompetent cells when preparing cells, pipetting out smaller parallel aliquots when transforming. In this way, transformation efficiencies of parallell samples during transforming should be very similiar. (If several smaller aliquots are used, aliquots may have a different history during preparation (f.ex: Come from different centrifugation tubes, experience differences during freezing process, etc.)


High efficiency transformation of Escherichia coli with plasmids:

One-step preparation of competent Escherichia coli:Transformation and storage of bacteria lcells in the same solution:

Die et al. 1982. Transformation In Escherichia coli: Studies On The Role Of The Heat Shock In Induction Of Competence:

Singh et al. International Journal of Biotechnology and Biochemistry ISSN 0973-2691 Volume 6 Number 4 (2010) pp. 561–568. Plasmid DNA Transformation in Escherichia Coli: Effect of Heat Shock Temperature, Duration, and Cold Incubation of CaCl2 Treated Cells:

High Efficiency 5 Min Transformation of Escherichia Coli:

C. T. CHUNG, SUZANNE L. NIEMELA, AND ROGER H. MILLER. 1989. One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution:

Compatibility groups

MICROBIOLOGICAL REVIEWS,Dec.1987,p. 381-395. Plasmid incompatibility:

Plasmid incompatibility: more compatible than previously thought?:



Cotransformation of linear chromosomal DNA and plasmid DNA in Escherichia coli:

Weston et al. 1979. Simultaneous transformation of Escherichia coli by pairs of compatible and incompatible plasmid DNA molecules:

Avoiding and controlling double transformation artifacts:

Natural transformaiton

Identification of a novel DNA element that promotes cell-to-cell transformation in Escherichia coli

Genome-wide screening of Escherichia coli genes involved in execution and promotion of cell-to-cell transfer of non-conjugative plasmids: RodZ (yfgA) is essential for plasmid acceptance in recipient cells

Natural DNA Uptake by Escherichia coli: