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From 2012.igem.org

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Revision as of 23:30, 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 Gel 4.1 RESULTS 5 V. QIAquick PCR Purification Protocol 6 VI. PCR Amplification of Cassette

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

• Make Gel:

• Measure out 1.00721 g of agarose and add to a 250 mL E-flask
• Add 50 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 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 TBE buffer

• Load Gel:

• Mix 3 samples (+2,+3,+4) in one microcentrifuge tube with 27 uL buffer. Load ~140 uL into one lane and the rest into another.
• Load on gel according to the following chart:

Lane 1	2	3-5
Invitrogen 1kb+ ladder	Cassette (~10 uL)	Cassette (~140 ul)

• Run Gel:

• Close gel box and turn on power pack
• Run gel at 30 V for ~1 hr, 20 min
• Run gel at 75 V until the markers have reached bottom of the gel

• Cut Gel:

• Remove gel from box and cut along lanes to separate the 10 uL lane from the 140 uL lane.
• Return 140 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 140 uL lane to the 10 uL lane
• Using the 10 uL lane as a guide, cut out the band from the 140 uL lane

II. QIAquick Gel Extraction Protocol

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

Cassette (+1)
485 mg

• Add 3 volumes Buffer QG to 1 volume of gel

Cassette (+1)
1455 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)
485 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
Cassette	(empty)

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

RESULTS

File: Media:srk_gel_extraction_2Aug12.xlsx

Concentration:

Sample	Concentration (ng/uL)	Approx. volume (uL)	Approx. total (ug)
Cassette	7.769	~28	0.22

IV. Image Post-Stained Gel

RESULTS

Source: Media:Srk_2012-08-02_18hr_08min.tif

Feature	Expected Size (bp)
Cassette	5152

V. QIAquick PCR Purification Protocol

• Add 5 volumes Buffer PB to 1 volume of reaction, mix

Reaction (+5)	Volume PB
50 uL	250 uL

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

• Apply sample to a QIAquick column, centrifuge for 1 minute at 13000 rpm, discard flow-through

• Add 750 uL of Buffer PE to the QIAquick column, centrifuge for 1 min and discard flow through

• Centrifuge again for 1 minute

• Transfer the column to clean microcentrifuge tube

• Elute DNA by adding 30 uL of Buffer EB to the center of the column and let stand for 1 minute

• Centrifuge for 1 minute, store DNA at 4 ^oC

VI. PCR Amplification of Cassette

• Mix Reagents:

• Mix according to the following table, where PP = PCR purified template and GE = gel extracted template

	-	PP 2	PP 5	PP 10	GE 2	GE 5	GE 10
Reagent	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)
water	33.5	31.5	28.5	23.5	31.5	28.5	23.5
5x Phusion HiFi buffer	10	10	10	10	10	10	10
10 mM dNTP mix	1	1	1	1	1	1	1
10 uM primer (F)	2.5 (pBf)	2.5 (pBf)	2.5 (pBf)	2.5 (pBf)	2.5 (pBf)	2.5 (pBf)	2.5 (pBf)
10 uM primer (R)	2.5 (pBr)	2.5 (pBr)	2.5 (pBr)	2.5 (pBr)	2.5 (pBr)	2.5 (pBr)	2.5 (pBr)
Template	0	2	5	10	2	5	10
Phusion HiFi polymerase	0.5	0.5	0.5	0.5	0.5	0.5	0.5

• Run PCR

Step	Temp (oC)	Time
1	94	4 m
2	94	30 s
3	66	30 s
4	72	1.5 m
10	72	7 m
11	4	∞

(left overnight)