Wittrup: Gel electrophoresis: Difference between revisions

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==Agarose Gel DNA Electrophoresis==
'''Materials'''
'''Materials'''


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* SYBR Gold DNA Stain (Invitrogen S-11494)
* SYBR Gold DNA Stain (Invitrogen S-11494)
* DNA gel box
* DNA gel box


''Restriction Stop Buffer (10x)''
''Restriction Stop Buffer (10x)''
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* 0. 015 g bromophenol blue
* 0. 015 g bromophenol blue
* to 15 mL ddH<sub>2</sub>O  
* to 15 mL ddH<sub>2</sub>O  


''Sample Loading Buffer (6x)''
''Sample Loading Buffer (6x)''
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* 0.025 g xylene cyanol
* 0.025 g xylene cyanol
* to 10 mL ddH<sub>2</sub>O
* to 10 mL ddH<sub>2</sub>O


'''Protocol'''  
'''Protocol'''  
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5. Pour slowly into case.  Allow gel to soldify (~30 min).
5. Pour slowly into case.  Allow gel to soldify (~30 min).


''Prepare Samples''  
''Prepare Samples''  
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DNA Digestion: 18 μL of restriction digested DNA, 2 μL 10x restriction stop buffer.
DNA Digestion: 18 μL of restriction digested DNA, 2 μL 10x restriction stop buffer.


''Complete and Run Gel''  
''Complete and Run Gel''  
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6. Allow the gel to run desired length.
6. Allow the gel to run desired length.
(bromophenol blue dye moves with 500 bp DNA)
(bromophenol blue dye moves with 500 bp DNA)


''Stain''  
''Stain''  
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5.  Incubate with rocking for >10 min.  Shield from light for long incubations.
5.  Incubate with rocking for >10 min.  Shield from light for long incubations.
(Stain may be reused if desired)
(Stain may be reused if desired)
== DNA Gel Extraction ==
'''Materials'''
QIAquick Gel Extraction kit (Qiagen 28706)
42-50˚C water bath
'''Protocol'''
This protocol is adapted/condensed from the Qiagen instructions.  The Qiagen site ([http://www1.qiagen.com/literature/handbooks/PDF/DNACleanupAndConcentration/QQ_Spin/1021422_HBQQSpin_072002WW.pdf]) offers several helpful tips that are useful for first-time users and for troubleshooting.
1. Using a razor blade, cut the desired piece from the gel and place in a microcentrifuge tube.
2. Add 3 gel volumes (often 300 μL for a small slice) of Buffer QG to the vial to dissolve agarose.
3. Incubate at 42-50˚C for 10 min. or until gel is fully dissolved.  Twice vortex briefly.
4. Upon complete dissolution, verify proper pH by checking for yellow color.
5. [Optional: only beneficial if DNA is <500 bp or >4000 bp] Add 1 gel volume of isopropanol to the vial and mix gently; do not centrifuge.
6. Apply sample to QIAquick column in a collection tube.
7. Centrifuge at 15,000-18,000g for 60 s and discard flowthrough.
8. Add 500 μL of Buffer QG to column to dissolve residual agarose.
9. Centrifuge at 15,000-18,000g for 60 s and discard flowthrough.
10. Add 750 μL of wash Buffer PE to column.
11. Centrifuge at 15,000-18,000g for 60 s and discard flowthrough.
12. Centrifuge at 15,000-18,000g for 60 s and discard flowthrough (to eliminate residual ethanol).
13. Place column in vial.
14. Add 30 μL of elution Buffer EB to center of column.
15. Let stand for 60 s.
16. Centrifuge at 15,000-18,000g for 60 s.
17. Store DNA collected in vial at -20˚C.

Latest revision as of 14:48, 6 March 2006

Materials

  • Restriction stop buffer (10x) or sample loading buffer (6x)
  • 10x TAE (Invitrogen 15528-026)
  • Agarose
  • DNA samples
  • DNA ladder: 1 kb (Invitrogen 15615-016) or 100 bp (Invitrogen 15628-019)
  • SYBR Gold DNA Stain (Invitrogen S-11494)
  • DNA gel box

Restriction Stop Buffer (10x)

  • 7.5 mL glycerol
  • 3 mL EDTA (0.5 M, pH 8.0)
  • 0.15 g sodium dodecylsulfate
  • 0. 015 g bromophenol blue
  • to 15 mL ddH2O

Sample Loading Buffer (6x)

  • 3 mL glycerol
  • 0.025 g bromophenol blue
  • 0.025 g xylene cyanol
  • to 10 mL ddH2O

Protocol

Prepare Gel

1. Prepare gel box: place tray with two side handles in rectangular gel box; add comb(s) with appropriate number of wells.

2. Add 50 mL of 1x TAE to a flask (this is for a small gel; larger gels require more).

3. Add appropriate amount of agarose (e.g., 0.50 g for a 1% gel).

4. Microwave flask until liquid boils (~40 s; watch carefully to avoid a spill). Swirl flask gently to mix.

5. Pour slowly into case. Allow gel to soldify (~30 min).

Prepare Samples

1. Combine ddH2O, buffer (either restriction stop buffer or loading buffer), and DNA.

Examples:

DNA Ladder: 0.5 μL of DNA ladder (1 μg/μL), ddH2O, 4.5 μL ddH2O, 1 μL 6x loading buffer

DNA Digestion: 18 μL of restriction digested DNA, 2 μL 10x restriction stop buffer.

Complete and Run Gel

1. When the gel has solidified, remove it from the gel box and place on upper level of electrophoresis apparatus. Remove comb.

2. Add 1x TAE to cover gel.

3. Pipette samples into the wells.

4. Cover case with red (-) electrode away from samples.

5. Apply constant voltage (70-100 V).

6. Allow the gel to run desired length. (bromophenol blue dye moves with 500 bp DNA)

Stain

Use caution: SYBR Gold binds DNA

1. Thaw SYBR Gold at room temperature (from 10 μL aliquot at -20º).

2. Add 40 mL of 1x TAE to container.

3. Add 10 μL of SYBR Gold to container. Mix well.

4. Place gel in container.

5. Incubate with rocking for >10 min. Shield from light for long incubations. (Stain may be reused if desired)

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