User:Jarle Pahr/Transformation: Difference between revisions
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=Commercial competent cells= | |||
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=Preparation of competent cells= | |||
http://forums.biotechniques.com/viewtopic.php?f=2&t=864 | http://forums.biotechniques.com/viewtopic.php?f=2&t=864 | ||
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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: | Significant variables in transformation: | ||
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(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.) | (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 | 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 | ||
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C. T. CHUNG, SUZANNE L. NIEMELA, AND ROGER H. MILLER. 1989. One-step preparation of competent Escherichia coli: Transformation | 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 | 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 |
Latest revision as of 08:35, 18 August 2013
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://johnson.cm.utexas.edu/kjohnson/methods/RapidTransformation.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 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. | http://www.addgene.org/plasmid_protocols/bacterial_transformation/ |
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 | 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.google.com/patents/US20020081565
http://johnson.cm.utexas.edu/kjohnson/methods/Inoue_Bacterial_Transformation.html
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