User:Jarle Pahr/Transformation

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Preparation of competent cells)
Line 74: Line 74:
Snap freezing may help avoid formation of ice crystals?
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.
 +
==Bibliography==
==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
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

Revision as of 11:46, 7 May 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://openwetware.org/wiki/IGEM:Paris_Bettencourt_2012/Protocols/Heat_Shock_Transformation_of_E_Coli


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 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?


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.


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

Personal tools