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From 2012.igem.org

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Revision as of 23:29, 3 October 2012

Contents 1 I. Prepare for Gel Extraction of a PCR Product from 27 Jul 2012 2 II. QIAquick Gel Extraction Protocol 3 III. Measure the Concentration of Extracted DNA 3.1 RESULTS 4 IV. Image Post-Stained Portion of Gel 4.1 RESULTS

I. Prepare for Gel Extraction of a PCR Product from 27 Jul 2012

• Make Gel:

• Measure out 0.75 g of agarose and add to a 250 mL E-flask
• Add 75 mL of 0.5x TBE buffer and swirl to mix
• Cover flask with a Kimwipe and microwave for ~1 minute until clear
• Allow to cool to ~60 ^oC
• Immediately pour gel into tray with combs
• 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 TBE buffer
• Note: Some gel leaked out the ends

• Load Gel:

• Mix sample in PCR tubes with 10 uL buffer. Load 40 uL into one lane and the rest into another.
• Load on gel according to the following chart:

Lane 1	2	3
Invitrogen 1kb+ ladder	+1 (~10 uL)	+1 (~40 ul)

• Run Gel:

• Close gel box and turn on power pack
• Run gel at 30 V for a while to stack gel (while at lunch)
• Run gel at 75 V until the markers have reached ~3/4 down the gel

• Cut Gel:

• Remove gel from box and cut along lanes to separate the 10 uL lane from the 40 uL lane.
• Return 40 uL lane 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
• Remove EtBr solution and wash with 200 mL of dH₂O for 10 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 40 uL lane to the 10 uL lane
• Using the 10 uL lane as a guide, cut out the band from the 40 uL lane

II. QIAquick Gel Extraction Protocol

• Weigh each gel slice in a colorless tube:

Cassette (+1)
389 mg

• Add 3 volumes Buffer QG to 1 volume of gel

Cassette (+1)
1167 uL

• Incubate in 50 ^oC water bath for 10-12 minutes until all gel has dissolved. Vortex to help mix.

• Note: The solutions were all yellow, OK to proceed

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

Cassette (+1)
389 uL

• Apply 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 columns and let the column stand for 5 minutes on the bench

• Centrifuge for 1 min and discard flow through

• Centrifuge again for 1 minute

• Transfer the columns to clean microcentrifuge tubes

• Elute DNA by adding 30 uL of Buffer EB to the center of the 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 (diverging, ignored)
Cassette	(empty)

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

RESULTS

File: Media:srk_gel_extraction_30jul12.xlsx

Concentration:

Sample	Concentration (ng/uL)	Approx. volume (uL)	Approx. total (ug)
Cassette	0	~28	0

IV. Image Post-Stained Portion of Gel

RESULTS

Source: Media:Srk_2012-07-30_15hr_02min.tif

Feature	Expected Size (bp)
Cassette	5152