Griffitts:Modified Eckhardt gel: Difference between revisions
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==Materials== | ==Materials== | ||
* [[Griffitts:Modified Eckhardt gel#20X SBE (500 mL)|SBE]] | * [[Griffitts:Modified Eckhardt gel#20X SBE (500 mL)|SBE]] | ||
* [[Griffitts:Modified Eckhardt gel#20 mg/mL RNase A (300 μL)|20 mg/mL RNase A]] | |||
* [[Griffitts:Modified Eckhardt gel# | |||
* [[Griffitts:Modified Eckhardt gel#0.3% Sarkosyl (1 mL)|0.3% Sarkosyl]] | * [[Griffitts:Modified Eckhardt gel#0.3% Sarkosyl (1 mL)|0.3% Sarkosyl]] | ||
* Sucrose | * Sucrose | ||
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* [[Griffitts:Common media#PH medium (1 L)|PH broth]] | * [[Griffitts:Common media#PH medium (1 L)|PH broth]] | ||
* [[Griffitts:Stock solutions#10% SDS (75 mL)|10% SDS]] | * [[Griffitts:Stock solutions#10% SDS (75 mL)|10% SDS]] | ||
* [[Griffitts:Stock solutions#Ethidium Bromide (1 mL)|1 mg/mL Ethidium Bromide]] | |||
* 1.7-mL microcentrifuge tubes | * 1.7-mL microcentrifuge tubes | ||
==Buffer preparation== | ==Buffer preparation== | ||
=== | ===20 mg/mL RNase A (300 μL)=== | ||
* | * 6 mg RNase A (in –20°C freezer) | ||
* | * 300 μL ddH<sub>2</sub>O | ||
* Vortex | * Vortex | ||
* Divide into 50 μL aliquots | * Divide into 50 μL aliquots | ||
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===20X SBE (500 mL)=== | ===20X SBE (500 mL)=== | ||
* 500 mL ddH<sub>2</sub>O | * 500 mL ddH<sub>2</sub>O | ||
* 4 g NaOH (=200 mM) | * 4 g NaOH (= 200 mM) | ||
* 3.72 g EDTA (=20 mM) | * 3.72 g EDTA (= 20 mM) | ||
* pH to 8.0 using boric acid (powder), ~18 g<br> | * pH to 8.0 using boric acid (powder), ~18 g<br> | ||
NOTE: Be sure to dilute this to 1X before using. | NOTE: Be sure to dilute this to 1X before using. | ||
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===SBE Lysis Buffer (1 mL)=== | ===SBE Lysis Buffer (1 mL)=== | ||
Prepare this fresh each time | |||
* 900 μL 1X [[Griffitts:Modified Eckhardt gel#20X SBE (500 mL)|SBE]] | * 900 μL 1X [[Griffitts:Modified Eckhardt gel#20X SBE (500 mL)|SBE]] | ||
* | * 100 mg sucrose (= 1%) | ||
* 10 μL [[Griffitts:Modified Eckhardt gel#100 mg/mL Lysozyme (1 mL)|100 mg/mL Lysozyme]] | * 10 μL [[Griffitts:Modified Eckhardt gel#100 mg/mL Lysozyme (1 mL)|100 mg/mL Lysozyme]] | ||
* | * 2 μL [[Griffitts:Modified Eckhardt gel#20 mg/mL RNase A (300 μL)|20 mg/mL RNase A]] | ||
Keep on ice | |||
==SBE gel preparation== | ==SBE mini-gel preparation== | ||
* 0.4 g agarose | * 0.4 g agarose | ||
* 50 mL 1X [[Griffitts:Modified Eckhardt gel#20X SBE (500 mL)|SBE]] | * 50 mL 1X [[Griffitts:Modified Eckhardt gel#20X SBE (500 mL)|SBE]] | ||
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** NOTE: Add the SDS right before pouring the gel, not before microwaving<br> | ** NOTE: Add the SDS right before pouring the gel, not before microwaving<br> | ||
** NOTE: Do '''not''' add ethidium bromide<br> | ** NOTE: Do '''not''' add ethidium bromide<br> | ||
NOTE: | ** NOTE: Pour the gel in the fridge and allow to set for 30 min. Don't let it go longer than that or the SDS will crash out of solution.<br> | ||
NOTE: Use | NOTE: Use a large-tooth comb | ||
== | ==Sample Preparation and Electrophoresis== | ||
* Grow bacteria in [[Griffitts:Common media# | * Grow bacteria overnight in [[Griffitts:Common media#LB broth (1 L)|LB]] | ||
* | * Subculture bacteria in [[Griffitts:Common media#LB broth (1 L)|LB]] and grow to an OD<sub>600</sub> of 0.6 | ||
* Put on ice | * Put on ice | ||
* | * Add 150 μL of each culture to 500 μL of chilled [[Griffitts:Modified Eckhardt gel#0.3% Sarkosyl (40 mL)|0.3% sarkosyl]] | ||
** If you have a culture with an OD<sub>600</sub> which does not equal 0.6, use the following shortcut: 90/OD<sub>600</sub> = the number of μL of culture to add to the sarkosyl | |||
* Centrifuge at maximum speed for 90 seconds | * Centrifuge at maximum speed for 90 seconds | ||
* Carefully pull off the supernatant | * Carefully pull off the supernatant | ||
** NOTE: Keep tubes on ice until you load them into the gel | |||
* Resuspend in 20 μL of [[Griffitts:Modified Eckhardt gel#SBE Lysis Buffer (1 mL)|SBE lysis buffer]] | * Resuspend in 20 μL of [[Griffitts:Modified Eckhardt gel#SBE Lysis Buffer (1 mL)|SBE lysis buffer]] | ||
** NOTE: Do '''not''' add DNA loading dye! | ** NOTE: Do '''not''' add DNA loading dye! | ||
* Immediately load into an [[Griffitts:Modified Eckhardt gel#SBE gel preparation|SBE gel]] | * Immediately load into an [[Griffitts:Modified Eckhardt gel#SBE mini-gel preparation|SBE mini-gel]] | ||
* Allow to sit | * Allow samples to sit in the well for 2 minutes. | ||
* Run at 23 V for | * Run at 23 V for 10 minutes (this is the lowest setting on our machines) | ||
* | ** NOTE: This works best if you run it without the lid | ||
* Increase the voltage to 100 V for 90 minutes | |||
==Staining== | ==Staining== | ||
* Place gel in 100 | * Place gel in 100 mL dH<sub>2</sub>O | ||
* Add 40 μL [[Griffitts:Stock solutions#Ethidium Bromide (1 mL)|ethidium bromide]] | * Add 40 μL [[Griffitts:Stock solutions#Ethidium Bromide (1 mL)|1 mg/mL ethidium bromide]] | ||
* Place on shaker at 50 rpm for 60 minutes | * Place on shaker at 50 rpm for 60 minutes | ||
* Discard ethidium bromide and replace with fresh dH<sub>2</sub>O | * Discard ethidium bromide and replace with fresh dH<sub>2</sub>O | ||
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<br> | <br> | ||
<br> | <br> | ||
'' | Adapted from '''Hynes, M. F. ''et al''.''' (1989) "Direct selection for curing and deletion of ''Rhizobium'' plasmids carrying the ''Bacillus subtilis sacB'' gene." ''Gene'' '''15:'''111–120. PMID 2548927. |
Latest revision as of 08:16, 20 April 2011
Materials
- SBE
- 20 mg/mL RNase A
- 0.3% Sarkosyl
- Sucrose
- 100 mg/mL Lysozyme
- PH broth
- 10% SDS
- 1 mg/mL Ethidium Bromide
- 1.7-mL microcentrifuge tubes
Buffer preparation
20 mg/mL RNase A (300 μL)
- 6 mg RNase A (in –20°C freezer)
- 300 μL ddH2O
- Vortex
- Divide into 50 μL aliquots
- Store at –20°C
100 mg/mL Lysozyme (1 mL)
- 100 mg lysozyme (in –20°C freezer)
- 1 mL ddH2O
- Vortex
- Divide into 100 μL aliquots
- Store at –20°C
20X SBE (500 mL)
- 500 mL ddH2O
- 4 g NaOH (= 200 mM)
- 3.72 g EDTA (= 20 mM)
- pH to 8.0 using boric acid (powder), ~18 g
NOTE: Be sure to dilute this to 1X before using.
0.3% Sarkosyl (40 mL)
- 40 mL 1X SBE
- 120 mg Sarkosyl
Note: Store at 4°C
SBE Lysis Buffer (1 mL)
Prepare this fresh each time
- 900 μL 1X SBE
- 100 mg sucrose (= 1%)
- 10 μL 100 mg/mL Lysozyme
- 2 μL 20 mg/mL RNase A
Keep on ice
SBE mini-gel preparation
- 0.4 g agarose
- 50 mL 1X SBE
- 2.5 mL 10% SDS
- NOTE: Add the SDS right before pouring the gel, not before microwaving
- NOTE: Do not add ethidium bromide
- NOTE: Pour the gel in the fridge and allow to set for 30 min. Don't let it go longer than that or the SDS will crash out of solution.
- NOTE: Add the SDS right before pouring the gel, not before microwaving
NOTE: Use a large-tooth comb
Sample Preparation and Electrophoresis
- Grow bacteria overnight in LB
- Subculture bacteria in LB and grow to an OD600 of 0.6
- Put on ice
- Add 150 μL of each culture to 500 μL of chilled 0.3% sarkosyl
- If you have a culture with an OD600 which does not equal 0.6, use the following shortcut: 90/OD600 = the number of μL of culture to add to the sarkosyl
- Centrifuge at maximum speed for 90 seconds
- Carefully pull off the supernatant
- NOTE: Keep tubes on ice until you load them into the gel
- Resuspend in 20 μL of SBE lysis buffer
- NOTE: Do not add DNA loading dye!
- Immediately load into an SBE mini-gel
- Allow samples to sit in the well for 2 minutes.
- Run at 23 V for 10 minutes (this is the lowest setting on our machines)
- NOTE: This works best if you run it without the lid
- Increase the voltage to 100 V for 90 minutes
Staining
- Place gel in 100 mL dH2O
- Add 40 μL 1 mg/mL ethidium bromide
- Place on shaker at 50 rpm for 60 minutes
- Discard ethidium bromide and replace with fresh dH2O
- Place on shaker at 50 rpm for 10 minutes
- Photograph under UV light
Adapted from Hynes, M. F. et al. (1989) "Direct selection for curing and deletion of Rhizobium plasmids carrying the Bacillus subtilis sacB gene." Gene 15:111–120. PMID 2548927.