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://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 |
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
