Miniprep/GET buffer: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
 
(42 intermediate revisions by 5 users not shown)
Line 1: Line 1:
Cheap non-kit plasmid miniprep:
<center>
'''A cheap, easy, and effective plasmid miniprep'''
</center>


# take 1.8 ml from an overnight culture into a 2 ml centrifuge tube, spin down 1 minute at full speed, and discard the medium
==Reagents==
# resuspend the bacterial pellet by vortexing with 100 ul of chilled (4C) GET solution
#GET buffer = 50 mM glucose (MW 180), 10mM EDTA, 25 mM Tris-HCl  pH 8
# lyse the bacteria by adding 200 ul of room temperature  alkaline SDS, not longer than 5 minutes.  Invert to mix: do not vortex.  The solution should become clear.
#Alkaline Buffer= 0.2 N NaOH
# precipitate proteins, membranes, and chromosomal DNA with 150 ul chilled (4C) potassium acetate solution, mix by inverting gently.
#Potassium acetate = 3 M potassium acetate, 1.8 M acetic acid, no pH adjustment
# centrifuge at full speed 5 to 10 min
#SDS
# transfer 400 ul of the clean supernatant into a new tube preloaded with 900 ul of 100% EtOH to precipitate the plasmid DNA
#PCA solution (optional) = 50 parts phenol, 49 parts chloroform, and 1 part amyl-alcohol
# place the tubes in the -80 freezer for 30 minutes
#Lysozyme (optional)
# centrifuge the precipitated plasmid DNA 10 to 15 minutes at full speed, and discard supernatant
# optional: wash the pellet with 70% EtOH, respin and discard the supernatant
# air dry the pellet for 10-15 minutes at room temperature with the open tube on the bench
# resuspend the plasmid DNA in 20 ul of TE.  The DNA will contain RNA contamination, which can be removed by resuspending in TE with RNAse.


==Procedure==
# Pipet 2 ml of overnight culture into a 2 ml centrifuge tube.
# Centrifuge for 1 minute at maximum speed and discard the supernatant.
# Add 100ul of refrigerated GET buffer to the pellet and vortex to resuspend.
# Freshly prepare the alkaline SDS solution by adding 10mg SDS per ml of 0.2M NaOH Solution.
# Add 200μl of alkaline SDS solution to the cell suspension and invert to mix. DO NOT VORTEX!  The solution should become clear. 
# Add 150ul of refrigerated potassium acetate solution and invert gently to mix. DO NOT VORTEX!  A precipitate should form.
# Store the tube on ice for 3-5 minutes. 
# Centrifuge for 10 minutes at maximum speed.
# Carefully pipet 400ul of the clean supernatant into a new tube.  DO NOT PICK UP ANY PRECIPITATE!!!
# Add 900ul of 100% (95% is ok too) EtOH to precipitate the plasmid DNA.
# Place the tubes in the -80 freezer for 30 minutes.
# Centrifuge the precipitated plasmid DNA 15 minutes at maximum speed (cold if possible)and discard supernatant.
# Carefully add 1ml 70% EtOH to the pellet and let sit for 3 minutes. 
# Centrifuge at maximum speed for 3 minutes.  Make sure the pellet is toward the outside.
# Discard the supernatant and air dry the pellet for 10-15 minutes.
# Once the pellet is COMPLETELY DRY resuspend the plasmid DNA in 20 ul of TE or distilled water.  The DNA will contain RNA contamination, which can be removed by resuspending in TE with RNAse.


GET buffer: 50 mM glucose, 10mM EDTA, 25 mM Tris-HCl  pH 8
==Optional Steps==


Alkaline SDS: 0.2 N NaOH, 1% SDS
*After step 3 add 10mg lysozyme and incubate for 30 mins before proceeding with step 5. This step is essential for lysing gram-positive cells.
*After step 9 add 400µl PCA solution to the tube, invert to mix, and centrifuge for 3 minutes at maximum speed.  Collect the upper phase by pipetting into a new tube and proceed with step 10.  This step helps remove any residual proteins.


Potassium acetate: 3 M potassium acetate, 1.8 M acetic acid, no pH adjustment
==Notes==
 
===Scale-up===
Even higher concentrations of plasmid have been obtained by scaling up this protocol 5x in 15ml centrifuge tubes. However, to do this a larger (i.e. slower) centrifuge was necessary to accomodate the larger tubes.  The adjusted centrifugation steps were as follows:</br>
::Step 9: Centrifuge at 5500g (Max speed for this larger centrifuge) for 15 minutes.</br>
::Step 13: Centrifuge at 5500g for 20 minutes.
 
==References==
 
This protocol is based on a protocol described by Sambrook and Russell in their manual "Molecular Cloning" (2001) p:1.32-1.34
 
==BioCoder version==
Following is the Miniprep/GET Buffer 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====
[[Miniprep - GET Buffer protocol]]
====Source Code====
[[Miniprep - GET Buffer protocol - source code]]
 
[[Category:Protocol]]
[[Category:DNA]]
[[Category:In vitro]]
[[Category:Escherichia coli]]

Latest revision as of 17:51, 13 January 2011

A cheap, easy, and effective plasmid miniprep

Reagents

  1. GET buffer = 50 mM glucose (MW 180), 10mM EDTA, 25 mM Tris-HCl pH 8
  2. Alkaline Buffer= 0.2 N NaOH
  3. Potassium acetate = 3 M potassium acetate, 1.8 M acetic acid, no pH adjustment
  4. SDS
  5. PCA solution (optional) = 50 parts phenol, 49 parts chloroform, and 1 part amyl-alcohol
  6. Lysozyme (optional)

Procedure

  1. Pipet 2 ml of overnight culture into a 2 ml centrifuge tube.
  2. Centrifuge for 1 minute at maximum speed and discard the supernatant.
  3. Add 100ul of refrigerated GET buffer to the pellet and vortex to resuspend.
  4. Freshly prepare the alkaline SDS solution by adding 10mg SDS per ml of 0.2M NaOH Solution.
  5. Add 200μl of alkaline SDS solution to the cell suspension and invert to mix. DO NOT VORTEX! The solution should become clear.
  6. Add 150ul of refrigerated potassium acetate solution and invert gently to mix. DO NOT VORTEX! A precipitate should form.
  7. Store the tube on ice for 3-5 minutes.
  8. Centrifuge for 10 minutes at maximum speed.
  9. Carefully pipet 400ul of the clean supernatant into a new tube. DO NOT PICK UP ANY PRECIPITATE!!!
  10. Add 900ul of 100% (95% is ok too) EtOH to precipitate the plasmid DNA.
  11. Place the tubes in the -80 freezer for 30 minutes.
  12. Centrifuge the precipitated plasmid DNA 15 minutes at maximum speed (cold if possible)and discard supernatant.
  13. Carefully add 1ml 70% EtOH to the pellet and let sit for 3 minutes.
  14. Centrifuge at maximum speed for 3 minutes. Make sure the pellet is toward the outside.
  15. Discard the supernatant and air dry the pellet for 10-15 minutes.
  16. Once the pellet is COMPLETELY DRY resuspend the plasmid DNA in 20 ul of TE or distilled water. The DNA will contain RNA contamination, which can be removed by resuspending in TE with RNAse.

Optional Steps

  • After step 3 add 10mg lysozyme and incubate for 30 mins before proceeding with step 5. This step is essential for lysing gram-positive cells.
  • After step 9 add 400µl PCA solution to the tube, invert to mix, and centrifuge for 3 minutes at maximum speed. Collect the upper phase by pipetting into a new tube and proceed with step 10. This step helps remove any residual proteins.

Notes

Scale-up

Even higher concentrations of plasmid have been obtained by scaling up this protocol 5x in 15ml centrifuge tubes. However, to do this a larger (i.e. slower) centrifuge was necessary to accomodate the larger tubes. The adjusted centrifugation steps were as follows:

Step 9: Centrifuge at 5500g (Max speed for this larger centrifuge) for 15 minutes.
Step 13: Centrifuge at 5500g for 20 minutes.

References

This protocol is based on a protocol described by Sambrook and Russell in their manual "Molecular Cloning" (2001) p:1.32-1.34

BioCoder version

Following is the Miniprep/GET Buffer 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

Miniprep - GET Buffer protocol

Source Code

Miniprep - GET Buffer protocol - source code

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Miniprep/GET_buffer&oldid=485260"