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Latest revision as of 01:47, 4 October 2012

Contents 1 I. Prepare for Gel Extraction of Cassette 2 II. QIAquick Gel Extraction Protocol 3 III. Measure the Concentration of Extracted DNA 3.1 RESULTS 4 IV. Image Post-Stained Gel 4.1 RESULTS

I. Prepare for Gel Extraction of Cassette

• Make Gel:

• Measure out 0.40668 g of agarose and add to a 250 mL E-flask
• Add 50 mL of 0.5x TBE buffer and swirl to mix (0.8% gel)
• Cover flask with a Kimwipe and microwave for ~1 minute until clear
• Allow to cool to ~60 ^oC
• Immediately pour gel into tray with modified combs (3 taped together for large well)
• Allow to solidify, then remove the comb and tape and place the tray in the gel box with the wells closer to the black electrode

• Make sure it is submerged in fresh TBE buffer

• Load Gel:

• Mix reactions 1 and 2 together and 3 and 4 together. Add 16 uL of 6x Loading Dye and mix. Ladder is premixed.

• Load on gel according to the following chart:

Lane 1	2	3-4	5	6	7-8
NEB 2-log ladder (4 uL)	PCRs 1/2 (~10 uL)	PCRs 1/2 (~86 ul)	NEB 2-log	PCRs 1/2 (~10 uL)	PCRs 1/2 (~86 ul)

• Run Gel:

• Close gel box and turn on power pack
• Run gel at 30 V for 30 min
• Run gel at 75 V until the markers have reached 3/4 of gel

• Cut Gel:

• Remove gel from box and cut along lanes to separate the 10 uL lanes from the 86 uL lanes.
• Return 86 uL lanes to the gel box

• Post-stain with EtBr

• Place 10 uL lane in 200 mL of 0.5 ug/mL EtBr solution for 15 minutes

• Image Gel

• View 10 uL lane under UV to identify band of interest
• Use a clean razor blade to nick gel where the band is

• Excise Gel Fragments

• Match 86 uL lanes to the 10 uL lanes
• Using the 10 uL lane as a guide, cut out the band from the 86 uL lane

II. QIAquick Gel Extraction Protocol

• Weigh each gel slice in a colorless 15 mL conical tube:

Cassette 1	Cassette 2
588 mg	520 mg

• Add 3 volumes Buffer QG to 1 volume of gel

Cassette 1	Cassette 2
1765 uL	1560 uL

• Incubate in 50 ^oC water bath for 12 minutes until all gel has dissolved. Vortex to help mix.

• Note: The solution was yellow, OK to proceed

• Add 1 volume 100% isopropanol to samples and mix by inversion

Cassette 1	Cassette 2
588 uL	588 uL <-- mistake

• Apply each sample to a QIAquick column 800 uL at a time. Centrifuge for 1 minute at 13000 rpm, discard flow-through, and repeat until all of the sample has passed through the column

• Add 500 uL of Buffer QG to the QIAquick columns and centrifuge for 1 minute, discard flow-through

• Add 750 uL of Buffer PE to the QIAquick column and incubate on the bench for 5 minutes

• Centrifuge for 1 min and discard flow through

• Centrifuge again for 1 minute

• Transfer the columns to clean microcentrifuge tubes

• Elute DNA by adding 50 uL of Buffer EB to the center of each column and let stand for 4 minutes

• Centrifuge for 1 minute

III. Measure the Concentration of Extracted DNA

• Bring sample and Buffer EB bottle upstairs to Take3 plate reader
• Add 2 uL of each sample according to the following chart:

Blank (EB)	Blank
Blank	Blank
1	2

• Use the program to blank the machine and measure sample concentrations

RESULTS

Concentration:

Sample	Concentration (ng/uL)	Approx. volume (uL)	Approx. total (ug)
Cassette 1	31.075	~48	1.49
Cassette 2	45.072	~48	2.16

IV. Image Post-Stained Gel

RESULTS

Source: Media:Srk_2012-08-09_14hr_49min.tif

Feature	Expected Size (bp)
Cassette	5152