Transforming chemically competent cells: Difference between revisions
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''Also see [[Preparing chemically competent cells]]'' | ''Also see [[Preparing chemically competent cells]]'' | ||
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#Incubate cells for 30 seconds at <math>42^o</math>C. | #Incubate cells for 30 seconds at <math>42^o</math>C. | ||
#*Note: According to the [http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid=2648393 original TSS paper] and qualitative experience ([[Josh Michener|JM]]), this step is completely optional and may actually reduce transformation efficiency. | #*Note: According to the [http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid=2648393 original TSS paper] and qualitative experience ([[Josh Michener|JM]]), this step is completely optional and may actually reduce transformation efficiency. | ||
#* I tested this with DH5a Z1 and pUC19 and found that heat shock at 42C for 30 sec improved transformation efficiency 10-fold ([[User:Paul_R_Jaschke|Paul Jaschke]]) | |||
#Incubate cells on ice for 2 min. | #Incubate cells on ice for 2 min. | ||
#Add 1 mL [[SOC]] ([[2XYT]] and LB are also suitable, original paper suggests LB + 20mM glucose) at room temp. | #Add 1 mL [[SOC]] ([[2XYT]] and LB are also suitable, original paper suggests LB + 20mM glucose) at room temp. | ||
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*'''[[User:Hugh Kingston|hugh kingston]] 09:46, 4 February 2008 (CST)''':I tried a few variations on this protocol, and found | *'''[[User:Hugh Kingston|hugh kingston]] 09:46, 4 February 2008 (CST)''':I tried a few variations on this protocol, and found | ||
*using SOC instead of LB + 20mM glucose increased efficiency 3 fold | *using SOC instead of LB + 20mM glucose increased efficiency 3 fold | ||
*heat shock of 42°C 45s increased efficiency 15-20 fold | *heat shock of 42°C 45s increased efficiency 15-20 fold compared to no heat shock | ||
==BioCoder version== | |||
Following is the Transforming chemically competent cells protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth|Vaishnavi]] | |||
====Text Output==== | |||
[[Transforming chemically competent cells protocol]] | |||
====Source Code==== | |||
[[Transforming chemically competent cells protocol - source code]] | |||
[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category:Escherichia coli]] | [[Category:Escherichia coli]] | ||
Latest revision as of 18:24, 16 May 2012
| back to protocols | ||
Also see Preparing chemically competent cells
Method
- Thaw TSS cells on ice.
- Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
- Note: If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
- Let sit for 30 minutes on ice.
- Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
- Incubate cells for 30 seconds at [math]\displaystyle{ 42^o }[/math]C.
- Note: According to the original TSS paper and qualitative experience (JM), this step is completely optional and may actually reduce transformation efficiency.
- I tested this with DH5a Z1 and pUC19 and found that heat shock at 42C for 30 sec improved transformation efficiency 10-fold (Paul Jaschke)
- Incubate cells on ice for 2 min.
- Add 1 mL SOC (2XYT and LB are also suitable, original paper suggests LB + 20mM glucose) at room temp.
- Incubate for 1 hour at [math]\displaystyle{ 37^o }[/math]C on shaker.
- Note: Can also save some time here by reducing incubation to ~45 min.
- Spread 100-300 μl onto a plate made with appropriate antibiotic.
- Grow overnight at 37 °C.
- Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.
- hugh kingston 09:46, 4 February 2008 (CST):I tried a few variations on this protocol, and found
- using SOC instead of LB + 20mM glucose increased efficiency 3 fold
- heat shock of 42°C 45s increased efficiency 15-20 fold compared to no heat shock
BioCoder version
Following is the Transforming chemically competent cells protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi
Text Output
Transforming chemically competent cells protocol
Source Code
Transforming chemically competent cells protocol - source code
