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Run Gel/ Gel extraction
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Latest revision as of 00:56, 30 October 2008
PRINCETON IGEM 2008
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Run Gel Protocol
Gel extractions are used to extract the DNA from the correct bands on a gel.
Preparing the Gel
Dissolve UltraPure agarose to a final concentration of 1% in TAE buffer in a glass bottle.
Heat the solution in the microwave with frequent stirring to dissolve the agarose homogenously.
Place the solution in a 55C water bath for 15 mins.
Add 1 µl EtBr (Ethidium Bromide) per 50 ml of the solution and mix well.
Pour 50ml of solution per small gel tray. (the gel trays and combs should be pre-cleaned with water and wiped dry).
Wait for the gels to solidify. Label and store at 4C.
Use 120ml per large gel tray. Insert one large and one small comb in each small gel tray.
Running the Gel
There are two kinds of wells that fit different volumes:
Small - 15 ul
Big - 40 ul
Appropriate Hyperladder to be used for PCR product which is linear. Usually Hyperladder I will be used.
1. Add gel loading buffer (Orange G 6X), add 1X to 5X of DNA (it helps DNA sink into the bottom of the well) to DNA.
2. Make sure there is enough 1xTAE in the plate holder.
3. Load 5ul of appropriate hyperladder to one of the lanes.
4. Load appropriate amount of DNA (mixed with the buffer) in each well.
5. Add 10ul Ethidium Bromide to 400 ml Buffer at the positive end (Approx. 1 ul EtBr per 50 ml Buffer).
6. Set the timer and voltage to 120V and 60 min.
Gel Extraction Protocol using QIAquick Gel Extraction Kit:
All centrifugation steps are done at speeds > 13000 rpm
1. Look at the gel under low wavelength UV (high wavelengths will denature DNA). Quickly take a polaroid image, cut out relevant bands and shut OFF the UV.
2. Place the cut bands in 2ml Eppendorf tubes; Weigh slices; No more than 400mg per tube
3. Add 3 volumes of Buffer QG to 1 volume of gel (100mg ~ 100ul)
4. Incubate at 50C for 10min or until gel is dissolved; vortex every 2-3 min
5. Confirm that color of mixture is yellow (if not, add 10ul of 3M NaAc, pH 5.0)
6. Add 1 gel volume of isopropanol (not necessary for DNA fragments between 500-4000bp)
7. Add max of 770ul to QIAquick column and centrifuge for 1 min (max speed, ~13,000rpm, RT)
8. Discard flow-through and place column back in tube.
9. If needed, add rest of mixture to same tube (up to additional 770ul), spin, and discard flow-through
10. Wash: add 0.75ml Buffer PE(make sure that the buffer has ethanol added to it) to column and centrifuge for 1 min
11. Discard flow-through & centrifuge for 1 min
12. Place column into clean Eppendorf tube
13. Add 50ul Buffer EB or water to center of membrane
14. Let stand at RT for 3 min
15. Centrifuge for 5 min
16. Measure the concentration using the UV spectrophotometer.
