User:Jarle Pahr/Transformation

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://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

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.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?

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

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