TITLE: Gel DNA Extraction 
AUTHORS: Kenneth Schackart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol details how to extract DNA from agarose gel using </span><a href="https://www.thermofisher.com/order/catalog/product/K0691#/K0691" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Thermo Scientific GeneJET Gel Extraction Kit</span></a><span>. This is suitable for gels run in either TAE or TBE buffer, and for DNA fragments from 25 bp to 20 kb. The extraction is performed using spin columns.</span></div></div>

[STEPS]
?. [Gel Electrophoresis]
Perform Gel Electrophoresis on your DNA sample to separate the DNA bands.
The protocol recommends    for TBE/agarose gel and    for TAE/agarose gel.Recommended well depth is 1 mm / 1 μL sample.Other recommendations can be found in the original protocol, such as recommended voltage.
?. [Gel Electrophoresis]
Stain gel for visualization.
?. [Excise Gel Bands]
Pre-weigh and mark 1.5 mL centrifuge tubes for each DNA band.
?. [Excise Gel Bands]
Weigh the tubes with gel, and determine gel mass.
?. [Excise Gel Bands]
Place gel in transilluminator, and using a clean scalpel or blade, closely excise the desired DNA band(s).
To avoid damage to the transilluminator surface, use a blunted blade.Each extraction column can process up to   1 agarose gel.
?. [Bind DNA to Column]
Add  Binding Buffer per   gel to centrifuge tube with gel (1:1 volume:weight).
100 µl
100 mg
For gels with greater than   agarose, use 2:1 volume:weight.
?. [Bind DNA to Column]
Incubate gel mixture at  until gel is completely dissolved. Agitate by inversion occasionally to improve mixing. Ensure that gel is completely dissolved before proceeding.
55 °C
?. [Bind DNA to Column]
Briefly vortex gel mixture.
?. [Bind DNA to Column]
Check color of solution. A yellow color is desired. If color is orange or violet, add  of  sodium acetate,   . Mix well.
10 µl
?. [Bind DNA to Column]
If DNA fragment length is ≤500 bp, add 1 gel volume of  isopropanol to gel solution. E.g.  isopropanol to  gel in  binding buffer. Mix well.
100 µl
100 mg
100 µl
?. [Bind DNA to Column]
Transfer up to  gel solution to extraction column.
800 µl
If gel solution volume exceeds   , add solution to column in stages; centrifuge for 1 min each time, discarding flow
?. [Bind DNA to Column]
Centrifuge at  for  .
Centrifuge: 14000 34
Always make a counter-balance when centrifuging. This is best achieved by weighing the tube and using water to make another tube of equal weight. Place same-weight pairs on opposite sides of centrifuge.A trick for getting the weight right quickly is to place tube with gel on balance, tare it, add empty tube to balance. Add   per   difference.
?. [Bind DNA to Column]
Discard flow through, place column back in same collection tube.
?. [Wash Column]
Add  wash buffer to purification column.
700 µl
?. [Wash Column]
Centrifuge at  for  . Discard flow through. Place column back in same collection tube.
Centrifuge: 14000 34
Be sure to make a new counter balance for this step and all later steps since the weights will change.
?. [Wash Column]
Centrifuge empty column at  for an additional  to remove any residual wash buffer. This step is not optional.
Centrifuge: 14000 34
?. [Elute DNA]
Transfer purification column to clean 1.5 mL centrifuge tube.
?. [Elute DNA]
Add  of Elution Buffer to the center of the column membrane.
50 µl
An elution volume between 20-50 μL can be used. Do not use less that   elution volume.
?. [Elute DNA]
Discard purification column. Store DNA at  .
-20 °C