Richard Lab:Agarose Gel Electrophoresis

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Back to [[Richard_Lab | Richard Lab]]
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Back to [[Richard_Lab:protocols | Protocols]]
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<center>'''This protocol is for the typical separation and purification of DNA done in the Richard Lab.'''  
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[[Image:Gel_electrophoresis.JPG|thumb|center|300px]]
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<br>'''The [[agarose gel electrophoresis | consensus electrophoresis protocol]] should be consulted if deviating from the procedure outlined here.'''</center>
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<center>
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'''This protocol is for the typical separation and purification of DNA done in the Richard Lab.   
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'''The [[agarose gel electrophoresis | consensus electrophoresis protocol]] should be consulted if using different reagents or otherwise deviating from the procedure outlined here.'''</center>
===Procedure===
===Procedure===
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# Place the gel tray perpendicular in the electrophoresis to create a casting bay.
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# Place the gel tray perpendicular in the electrophoresis chamber to create a casting bay.
# Wipe the tray clean with a Kim-wipe and level the tray using the bubble level.
# Wipe the tray clean with a Kim-wipe and level the tray using the bubble level.
# Weigh out the desired amount of agarose and add it to 100ml of 1X TBE in a 300ml flask ([[User:Michael_A. Speer | Mike]] usually uses 1.3 - 1.4g).
# Weigh out the desired amount of agarose and add it to 100ml of 1X TBE in a 300ml flask ([[User:Michael_A. Speer | Mike]] usually uses 1.3 - 1.4g).
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## 10ul DNA ladder
## 10ul DNA ladder
## 1ul [[SYBR_Green_II|SYBR green]] (100X)
## 1ul [[SYBR_Green_II|SYBR green]] (100X)
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## 1ul Bromophenol Blue
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## 1ul [[Agarose_gel_loading_dye|Bromophenol Blue]]
# Prepare the DNA Samples by combining the following:
# Prepare the DNA Samples by combining the following:
## 40ul DNA preparation
## 40ul DNA preparation
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===Notes===
===Notes===
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* Our "1X TBE" is technically 0.5X TBE but for our purposes we call it 1X TBE.
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* Our "1X TBE" is technically 0.5X TBE (5.3g/L Tris Base, 2.75g/L Boric Acid, and 2.9g/L EDTA) but for our purposes we call it 1X TBE.
** Correspondingly, the 10X TBE is officially 5X.
** Correspondingly, the 10X TBE is officially 5X.
*Using TBE allows us to have such a high (150V) voltage.  If you use TAE you need to use a significantly lower voltage and your run time will be longer.  Despite that, TAE is advantagous in some cases, but [[User:Michael_A. Speer | Mike]] feels that TBE is better suited for his applications.  This is discussed in detail in the [[agarose gel electrophoresis | consensus  protocol]].  
*Using TBE allows us to have such a high (150V) voltage.  If you use TAE you need to use a significantly lower voltage and your run time will be longer.  Despite that, TAE is advantagous in some cases, but [[User:Michael_A. Speer | Mike]] feels that TBE is better suited for his applications.  This is discussed in detail in the [[agarose gel electrophoresis | consensus  protocol]].  
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* Use the following table to determine the amount of agarose you want to use.  
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* Use the following table to determine the amount of agarose you want to use.<br><br>
<center>
<center>
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{| border="1" cellpadding="5" cellspacing="0" align="center"  
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{| border="1" cellpadding="5" cellspacing="0" align="center"  
|+ '''Choosing an Agarose Concentration'''
|+ '''Choosing an Agarose Concentration'''
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! Agarose Concentration(g/100mL) !! Optimal DNA Resolution (kb)  
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! Agarose Concentration (g/100mL) !! Optimal DNA Resolution (kb)  
|-=
|-=
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| 0.5 || 1 - 30
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| align="center"|0.5 || align="center"|1 - 30
|-
|-
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| 0.7 || 0.8 - 12
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| align="center"|0.7 || align="center"|0.8 - 12
|-
|-
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| 1.0 || 0.5 - 10
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| align="center"|1.0 || align="center"|0.5 - 10
|-
|-
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| 1.2 || 0.4 - 7
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| align="center"|1.2 || align="center"|0.4 - 7
|-
|-
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| 1.5 || 0.2 - 3
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| align="center"|1.5 || align="center"|0.2 - 3
|}
|}
</center>
</center>
[[Category:Protocol]] [[Category:In vitro]] [[Category:DNA]]
[[Category:Protocol]] [[Category:In vitro]] [[Category:DNA]]
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Back to [[Richard_Lab | Richard Lab]]
+
Back to [[Richard_Lab:protocols | Protocols]]

Current revision

Back to Protocols

This protocol is for the typical separation and purification of DNA done in the Richard Lab.

The consensus electrophoresis protocol should be consulted if using different reagents or otherwise deviating from the procedure outlined here.

Procedure

  1. Place the gel tray perpendicular in the electrophoresis chamber to create a casting bay.
  2. Wipe the tray clean with a Kim-wipe and level the tray using the bubble level.
  3. Weigh out the desired amount of agarose and add it to 100ml of 1X TBE in a 300ml flask ( Mike usually uses 1.3 - 1.4g).
  4. Microwave the flask on high for 80 seconds. Be sure that it does not boil over.
  5. Swirl the microwaved agarose in the flask until the solution becomes clear.
  6. Pour the melted solution into the casting bay and insert the comb.
  7. Prepare the DNA ladder by combining the following:
    1. 10ul DNA ladder
    2. 1ul SYBR green (100X)
    3. 1ul Bromophenol Blue
  8. Prepare the DNA Samples by combining the following:
    1. 40ul DNA preparation
    2. 4ul SYBR Green (100X)
    3. 4ul Bromophenol Blue
  9. Remove the comb from the cured gel and realign the gel in the chamber.
  10. Adjust the buffer level by adding 1X TBE to the chamber until the buffer just covers the gel. The buffer can be reused a few times.
  11. Pipette the samples into the wells
  12. Apply 150 volts and run for approximately 60 minutes.
  13. Photograph the gel using the UVP transilluminator system.

Notes

  • Our "1X TBE" is technically 0.5X TBE (5.3g/L Tris Base, 2.75g/L Boric Acid, and 2.9g/L EDTA) but for our purposes we call it 1X TBE.
    • Correspondingly, the 10X TBE is officially 5X.
  • Using TBE allows us to have such a high (150V) voltage. If you use TAE you need to use a significantly lower voltage and your run time will be longer. Despite that, TAE is advantagous in some cases, but Mike feels that TBE is better suited for his applications. This is discussed in detail in the consensus protocol.
  • Use the following table to determine the amount of agarose you want to use.

Choosing an Agarose Concentration
Agarose Concentration (g/100mL) Optimal DNA Resolution (kb)
0.5 1 - 30
0.7 0.8 - 12
1.0 0.5 - 10
1.2 0.4 - 7
1.5 0.2 - 3

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