Chromosomal DNA isolation from E. coli: Difference between revisions
(New page: ==Curators== '''~~~~''' ''Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare com...) |
m (→Procedure) |
||
| (9 intermediate revisions by 4 users not shown) | |||
| Line 1: | Line 1: | ||
{{back to protocols}} | |||
==Curators== | ==Curators== | ||
'''[[User:Torsten Waldminghaus|Torsten Waldminghaus]] | '''[[User:Torsten Waldminghaus|Torsten Waldminghaus]]''' | ||
''Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare community is currently [[OpenWetWare:Information management/Protocol curators|discussing the idea of protocol curators]]. Please contribute.'' | ''Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare community is currently [[OpenWetWare:Information management/Protocol curators|discussing the idea of protocol curators]]. Please contribute.'' | ||
| Line 9: | Line 11: | ||
==Materials== | ==Materials== | ||
*10% SDS | *10% SDS | ||
* | *Isopropanol | ||
* | *Ethanol | ||
* | *3 M NaOAc | ||
===Reagents=== | ===Reagents=== | ||
*[[TE]] | |||
*[[Killing Buffer]] | |||
===Equipment=== | ===Equipment=== | ||
*water bath at 65°C | |||
==Procedure== | ==Procedure== | ||
* grow culture in your favorite medium | |||
* Mix samples directly with ice cold [[Killing Buffer]] in ratio 1:1 and put on ice (samples should be processed as fast as possible) | |||
* Spin down cells 3 min max. speed at 4°C and discard supernatant | |||
* resuspend in 300 μL [[TE]] and add 40 μL 10% SDS and 3 μL 0.5 M EDTA | |||
[[ | * incubate 5 min at 65°C | ||
* add 750 μL isopropanol and mix | |||
[[ | * spin at max. speed for 5 min at room temperature and discard supernatant | ||
* resuspend pellet in 500 μL TE and add 2 μL RNase A (25 mg/ml) | |||
* incubate for 30 min at 65°C | |||
* add 2 μL [[proteinase K]] (25 mg/ml) and incubate at 37°C for 15 min | |||
[[ | * phenol extract (2x phenol & 2x chlorophorm) | ||
* precipitate over night with 1 ml ethanol and 40 μL 3M Na-Acetate | |||
* spin down DNA at 4°C for 15 min, wash in 70% ethanol and resuspend pellet in 50 μL dH<sub>2</sub>O | |||
==Critical steps== | ==Critical steps== | ||
| Line 43: | Line 47: | ||
Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br> | Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br> | ||
==BioCoder version== | |||
Following is the Chromosomal DNA isolation from E. coli 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==== | ||
[[Chromosomal DNA isolation from E. coli protocol]] | |||
====Source Code==== | |||
[[Chromosomal DNA isolation from E. coli protocol - source code]] | |||
== | |||
== | |||
[[Category:Protocol]] | [[Category:Protocol]][[Category:Needs attention]] | ||
Latest revision as of 08:16, 31 July 2012
| back to protocols | ||
Curators
Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare community is currently discussing the idea of protocol curators. Please contribute.
Abstract
This protocol describes the isolation of chromosomal DNA from E. coli cells. The DNA could be used for Southern Blots, as template for PCR or for DNA microarrays.
Materials
- 10% SDS
- Isopropanol
- Ethanol
- 3 M NaOAc
Reagents
Equipment
- water bath at 65°C
Procedure
- grow culture in your favorite medium
- Mix samples directly with ice cold Killing Buffer in ratio 1:1 and put on ice (samples should be processed as fast as possible)
- Spin down cells 3 min max. speed at 4°C and discard supernatant
- resuspend in 300 μL TE and add 40 μL 10% SDS and 3 μL 0.5 M EDTA
- incubate 5 min at 65°C
- add 750 μL isopropanol and mix
- spin at max. speed for 5 min at room temperature and discard supernatant
- resuspend pellet in 500 μL TE and add 2 μL RNase A (25 mg/ml)
- incubate for 30 min at 65°C
- add 2 μL proteinase K (25 mg/ml) and incubate at 37°C for 15 min
- phenol extract (2x phenol & 2x chlorophorm)
- precipitate over night with 1 ml ethanol and 40 μL 3M Na-Acetate
- spin down DNA at 4°C for 15 min, wash in 70% ethanol and resuspend pellet in 50 μL dH2O
Critical steps
Referenced from the main protocol, a more thorough explanation of particularly important steps in the protocol.
Troubleshooting
Referenced from the main protocol, an explanation of what can cause things to go wrong with the protocol.
Notes
Referenced from the main protocol, any comments about the protocol should be made here; i.e. how it was developed. Any comments added should be signed (by adding *'''~~~~''': in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.
Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.
BioCoder version
Following is the Chromosomal DNA isolation from E. coli 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
Chromosomal DNA isolation from E. coli protocol
Source Code
Chromosomal DNA isolation from E. coli protocol - source code
