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-	''File:'' [[Media:srk_gel_extraction_15aug12.xlsx]]
	''Concentration:''		''Concentration:''

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Latest revision as of 01:41, 4 October 2012

Contents 1 I. Measure the Concentration of Extracted DNA 1.1 RESULTS 2 II. Ligate Digested Plasmid and Digested Cassette Together 3 III. Prepare for Gel Extraction of a PCR Product from 14 Aug 2012 4 IV. QIAquick Gel Extraction Protocol 5 V. Measure the Concentration of Extracted DNA 5.1 RESULTS 6 VI. Image Post-Stained Gel 6.1 RESULTS

I. Measure the Concentration of Extracted DNA

• Bring samples and water upstairs to Take3 plate reader
• Add 2 uL of each sample according to the following chart:

Blank (water)	Blank
Blank	Blank
Digested Plasmid	Digested Cassette

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

RESULTS

Concentration:

Sample	Concentration (ng/uL)	Approx. volume (uL)	Approx. total (ug)
Digested Plasmid	13.031	~28	0.365
Digested Cassette	13.021	~28	0.365

II. Ligate Digested Plasmid and Digested Cassette Together

• 1:1 vector:insert ratio - Mix:

• 2 uL 10X T4 Buffer
• 6 uL plasmid DNA (~78 ng)
• 11 uL cassette DNA (~143 ng)
• 1 uL T4 Ligase

• 1:2 vector:insert ratio - Mix:

• 2 uL 10X T4 Buffer
• 3.64 uL plasmid DNA (~47 ng)
• 13.36 uL cassette DNA (~174 ng)
• 1 uL T4 Ligase

[http://molbiol.edu.ru/eng/scripts/h01_07.html This website] was helpful

• Incubate at room temperature for 2 hours, then David will use it to transform cells

III. Prepare for Gel Extraction of a PCR Product from 14 Aug 2012

• Make Gel:

• Measure out 0.40745 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 (2 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 4 samples (C1-4) in one tube with 20 uL loading dye. Load ~10 uL into one lane and the rest into another.
• Load on gel according to the following chart:

Lane 1	2	3-4
NEB 2-log ladder (4 uL, premixed)	Cassette (~10 uL)	Cassette (~110 ul)

• Run Gel:

• Close gel box and turn on power pack
• Run gel at 30 V for 26 min
• Run gel at 75 V until the markers have reached near bottom of the gel

• Cut Gel:

• Remove gel from box and cut along lanes to separate the 10 uL lane from the 110 uL lane.
• Return 110 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

• 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 110 uL lane to the 10 uL lane
• Using the 10 uL lane as a guide, cut out the band from the 110 uL lane

IV. QIAquick Gel Extraction Protocol

• Weigh each gel slice in a colorless microcentrifuge tube:

Cassette
376 mg

• Add 3 volumes Buffer QG to 1 volume of gel

Cassette (+1)
1128 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)
376 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, 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

V. 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

Concentration:

Sample	Concentration (ng/uL)	Approx. volume (uL)	Approx. total (ug)
Cassette	27.065	~28	0.76

VI. Image Post-Stained Gel

RESULTS

Source: Media:Srk_2012-08-15_17hr_55min.tif

Feature	Expected Size (bp)
Cassette	5152