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
Kanamycin works by interfering with protein translation.
High efficiency transformation of Escherichia coli with plasmids: http://www.sciencedirect.com/science/article/pii/037811199090336P
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
Snap freezing may help avoid formation of ice crystals?
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.)
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