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: http://www.protocol-online.org/biology-forums/posts/39293.html

See also http://www.protocol-online.org/biology-forums/posts/24469.html

Kanamycin works by interfering with protein translation.

http://www.genome.ou.edu/protocol_book/protocol_adxF.html

http://www.protocol-online.org/biology-forums/posts/25521.html

http://www.ncbi.nlm.nih.gov/pubmed/12065899

http://bitesizebio.com/articles/plasmid-retention/

http://www.protocol-online.org/biology-forums/posts/24469.html

http://openwetware.org/wiki/IGEM:Paris_Bettencourt_2012/Protocols/Heat_Shock_Transformation_of_E_Coli

http://johnson.cm.utexas.edu/kjohnson/methods/RapidTransformation.html

http://www.sigmaaldrich.com/life-science/molecular-biology/cloning-and-expression/learning-center/transformation-efficiency.html

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC145660/

http://2010.igem.org/wiki/images/a/ae/Direct_Plating_Transformation_Protocol.pdf


High efficiency transformation of Escherichia coli with plasmids: http://www.sciencedirect.com/science/article/pii/037811199090336P

https://www.neb.com/protocols/2012/05/21/transformation-protocol

https://www.neb.com/protocols/1/01/01/5-minute-transformation-protocol-c2987

http://sheatechinc.com/Ligation_Transformation.html

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. http://partsregistry.org/Help:Protocols/Transformation
AddGene 20-50 uL1-5 uL (10 pg to 100 ng) 20-30 min 42 C 30-60 sOn ice for 2 min. Add 200-250 uL SOC, 37 C for 45 min. http://www.addgene.org/plasmid_protocols/bacterial_transformation/


Contents

Commercial competent cells

Name Description/Use Supplier (Main) Genotypes Transformation efficiency (cfu/ug) Comment Reference
XL10-GOLD Ultracompetent cellsChemocompetent cellsStratageneHte>5x10^9 http://www.freewebs.com/labjuan2/XL10%20competent%20cells%20STRATAGENE.pdf

Preparation of competent cells

http://forums.biotechniques.com/viewtopic.php?f=2&t=864

http://mcb.berkeley.edu/labs/krantz/protocols/calcium_comp_cells.pdf

http://www.protocol-online.org/biology-forums/posts/7508more2.html


http://www.princeton.edu/genomics/mcclean/protocols/Inoue-Method-for-Preparation-of-Competent-E-Coli.pdf

http://www.google.com/patents/US20020081565

http://johnson.cm.utexas.edu/kjohnson/methods/Inoue_Bacterial_Transformation.html

http://groselab.byu.edu/Portals/92/Protocols%20for%20Andrew/Basic%20lab%20protocols/DH5%20alpha%20Competent%20Cells.doc

Snap freezing may help avoid formation of ice crystals?


Inoue 1990.: http://www.ncbi.nlm.nih.gov/pubmed/2265755


Chung et al.: http://www.pnas.org/content/86/7/2172.full.pdf

Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors.: http://www.ncbi.nlm.nih.gov/pubmed/20027870

Transformation

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.)

Bibliography

High efficiency transformation of Escherichia coli with plasmids: http://www.sciencedirect.com/science/article/pii/037811199090336P


One-step preparation of competent Escherichia coli:Transformation and storage of bacteria lcells in the same solution: http://www.pnas.org/content/86/7/2172.full.pdf

Die et al. 1982. Transformation In Escherichia coli: Studies On The Role Of The Heat Shock In Induction Of Competence: http://mic.sgmjournals.org/content/129/3/663


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: http://www.itescam.edu.mx/principal/sylabus/fpdb/recursos/r72068.PDF


High Efficiency 5 Min Transformation of Escherichia Coli: http://nar.oxfordjournals.org/content/24/3/536.full


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: http://www.pnas.org/content/86/7/2172.full.pdf

http://www.ejbiotechnology.info/content/vol10/issue1/full/10/index.html

Compatibility groups

MICROBIOLOGICAL REVIEWS,Dec.1987,p. 381-395. Plasmid incompatibility: http://europepmc.org/articles/PMC373122/pdf/microrev00051-0011.pdf


Plasmid incompatibility: more compatible than previously thought?: http://peds.oxfordjournals.org/content/20/7/309.full


IncP

http://www.annualreviews.org/doi/pdf/10.1146/annurev.mi.41.100187.000453

Co-transformation

http://www.biomedcentral.com/1472-6750/10/56

Cotransformation of linear chromosomal DNA and plasmid DNA in Escherichia coli: http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.1980.tb05638.x/abstract


Weston et al. 1979. Simultaneous transformation of Escherichia coli by pairs of compatible and incompatible plasmid DNA molecules: http://link.springer.com/article/10.1007%2FBF00276222

http://bitesizebio.com/articles/plasmid-retention/


Avoiding and controlling double transformation artifacts: http://peds.oxfordjournals.org/content/20/7/315.full


Natural transformaiton

Identification of a novel DNA element that promotes cell-to-cell transformation in Escherichia coli http://www.sciencedirect.com/science/article/pii/S0014579311004066


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: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035620

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