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From 2012.igem.org

Contents 1 I. Run Diagnostic PCR on Sample 3 2 II. Prepare for Gel Extraction of PCR Products from 21 Jul 2012 3 III. QIAquick Gel Extraction Protocol 4 IV. Measure the Concentration of Extracted DNA 4.1 RESULTS 5 V. AGE of Diagnostic PCR samples

I. Run Diagnostic PCR on Sample 3

• Mix Reagents:

• Mix according to the following table

	A	B	C	D	E	F
Reagent	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)
water	33.5	34.5	36	36	33.5	34
5x Phusion HiFi buffer	10	10	10	10	10	10
10 mM dNTP mix	1	0	1	1	1	1
10 uM primer (F)	2.5 (p3f_2)	2.5 (p3f_2	0	2.5 (p3f_2)	2.5 (p3f_2)	2.5 (p3f_2)
10 uM primer (R)	2.5 (p3r)	2.5 (p3r)	2.5 (p3r)	0	2.5 (p3r)	2.5 (p3r)
Template	ECNR2 colony*	ECNR2 colony*	ECNR2 colony*	ECNR2 colony*	0	ECFI5 colony*
Phusion HiFi polymerase	0.5	0.5	0.5	0.5	0.5	0

*For these templates, I touched a colony with a sterile pipette tip and spread it on the bottom of the PCR tube before adding reagents

• Run PCR

Step	Temp (oC)	Time
1	94	4 m
2	94	30 s
3	53	30 s
4	72	1.5 m
5	GOTO 2	7x
6	94	30 s
7	66	30 s
8	72	1.5 m
9	GOTO 6	30x
10	72	5 m
11	4	∞*

• Store tubes on ice after PCR is finished

II. Prepare for Gel Extraction of PCR Products from 21 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

• Load Gel:

• Mix samples in PCR tubes with 10 uL buffer. Load 40 uL into one lane and the rest into another.
• Load ~6 uL on gel according to the following chart:

Lane 1	2	3	4	5	6	7	8
NEB 2-log ladder	2.2 (~10 uL)	2.2 (~40 ul)	NEB 2-log ladder	3.2 (~10 uL)	3.2 (~40 uL)	4.1 (~10 uL)	4.1 (~40 uL)

• Store DNA at 4 ^oC

• Run Gel:

• Close gel box and turn on power pack
• Run gel at 30 V for 25 min to stack gel
• 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 lanes from the 40 uL lanes.
• Return 40 uL lanes to the gel box

• Post-stain with EtBr

• Place 10 uL lanes 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 lanes under UV to identify bands of interest
• Use a clean razor blade to nick gel where the bands are

• Excise Gel Fragments

• Match 40 uL lanes to the 10 uL lanes
• Using the 10 uL lanes as a guide, cut out the bands from the 40 uL lanes

III. QIAquick Gel Extraction Protocol

• Weigh each gel slice in a colorless tube:

LacZ (2.2)	CAT* (3.2)	tetr <4.1)
233 mg	263 mg	409 mg (treated as 400 mg)

• Add 3 volumes Buffer QG to 1 volume of gel

LacZ (2.2)	CAT* (3.2)	tetr <4.1)
699 uL	789 uL	1200 uL

• Incubate in 50 ^oC water bath for 10-15 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

LacZ (2.2)	CAT* (3.2)	tetr <4.1)
233 uL	263 uL	409 uL

• 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 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 5 minutes

• Centrifuge for 1 minute

IV. Measure the Concentration of Extracted DNA

• Bring samples 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)
LacZ	CAT*
tetR	(empty)

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

RESULTS

Concentration:

Plasmid	Concentration (ng/uL)	Approx. volume (uL)	Approx. total (ug)
LacZ	11.468	~28	0.321
CAT*	35.256	~28	0.987
tetR	37.787	~28	1.058

V. AGE of Diagnostic PCR samples

• Make Gel:

• Measure out 0.5 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 and pour into flask for ethidium bromide (EtBr)
• Add 1 uL of EtBr stock to agarose and swirl to mix
• 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 fresh TBE buffer

• Load Gel:

• Mix samples as drops on a piece of parafilm

• Mix:

Ladder (NEB 2-log)	Blank	Sample
*5 uL DNA ladder *1 uL 6x Loading Dye	*8.3 uL dH2O *1.7 uL 6x Loading Dye	*6.3 uL dH2O *1.7 uL 6x Loading Dye *2.0 uL DNA

• Load ~8 uL (except ladder which is ~6 uL) on gel according to the following chart:

Lane 1	2	3	4	5	6	7	8	9	10	11
NEB 2-log ladder	empty	Blank	A	empty	B	empty	C	D	E	F

• Store DNA at 4 ^oC
• Unusual loading pattern was due to poor loading

• Run Gel:

• Close gel box and turn on power pack
• Run gel at 30 V for 20 min to stack gel
• Run gel at 75 V until the markers have reached ~halfway down gel

• Image Gel:

• RESULTS:

Source: Media:Srk_2012-07-23_21hr_46min.tif