"

From 2012.igem.org

Contents 1 I. Prepare for Gel Extraction of LacZ Product from HF2 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. Cross-over PCR 6 VI. AGE of PCR Samples

I. Prepare for Gel Extraction of LacZ Product from HF2

• Make Gel:

• Measure out 0.40175 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:

Ladder (NEB 2-log)	10 uL well	42 uL well
*4 uL pre-mixed	*6.3 uL dH2O *1.7 uL 6x Loading Dye *2.0 uL DNA	*6 uL 6x Loading Dye *36 uL DNA

• Load on gel according to the following chart:

Lane 1	2	3-5
NEB 2-log ladder	LacZ (~10 uL)	LacZ (~45 ul)

• Run Gel:

• Close gel box and turn on power pack
• Run gel at 30 V for 20 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 lane from the 45 uL lane.
• Return 45 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 45 uL lane to the 10 uL lane
• Using the 10 uL lane as a guide, cut out the band from the 45 uL lane

II. QIAquick Gel Extraction Protocol

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

LacZ (HF2)
275 mg

• Add 3 volumes Buffer QG to 1 volume of gel

LacZ (HF2)
825 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

LacZ (HF2)
275 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 column and centrifuge for 1 minute, 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 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
LacZ	(empty)

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

RESULTS

File: Media:srk_gel_extraction_8aug12.xlsx

Concentration:

Sample	Concentration (ng/uL)	Approx. volume (uL)	Approx. total (ug)
LacZ	77.185	~28	2.16

IV. Image Post-Stained Gel

RESULTS

Source: Media:Srk_2012-08-08_13hr_42min.tif

Feature	Expected Size (bp)
LacZ	3151

V. Cross-over PCR

• Template Mix Specifications:

Mix I:

Template	Size (bp)	Amount Wanted (pmol)	Amount Wanted (ng)	Concentration (ng/uL)	Volume (1x)	Volume (5x)
LacZ	3151	0.01	20.45	77.185	0.26	1.32
CAT*	729	0.01	4.73	35.256	0.13	0.67
tetR	1265	0.01	8.21	37.787	0.22	1.09
total						3.08 uL

Mix II:

Template	Size (bp)	Amount Wanted (pmol)	Amount Wanted (ng)	Concentration (ng/uL)	Volume (1x)	Volume (5x)
LacZ	3151	0.05	102.25	77.185	1.32	6.62
CAT*	729	0.05	23.65	35.256	0.67	3.35
tetR	1265	0.05	41.05	37.787	1.09	5.45
total						15.42 uL

• *Calculations made with [http://molbiol.edu.ru/eng/scripts/h01_07.html this website]

• Mix Reagents:

• Mix according to the following table

Mix I:

	1 Reaction	Master Mix (x6)
Reagent	(uL)	(uL)
water	32.384	194.3
5x KAPA HiFi buffer	10	60
10 mM dNTP mix	1.5	9
10 uM primer (F)	2.5 (pBf)	15 (pBf)
10 uM primer (R)	2.5 (pBr)	15 (pBr)
Template Mix	0.616	-
KAPA HiFi polymerase	0.5	3

Mix II:

	1 Reaction	Master Mix (x6)
Reagent	(uL)	(uL)
water	29.916	179.496
5x KAPA HiFi buffer	10	60
10 mM dNTP mix	1.5	9
10 uM primer (F)	2.5 (pBf)	15 (pBf)
10 uM primer (R)	2.5 (pBr)	15 (pBr)
Template Mix	3.084	-
KAPA HiFi polymerase	0.5	3

• Before adding template, move 49.384 uL of Master Mix I to a clean PCR tube and add 0.616 uL water for negative control
• Before adding template, move 46.916 uL of Master Mix II to a clean PCR tube and add 3.084 uL water for negative control
• Add templates to Master Mixes according to Template Specifications above, mix by pipetting
• Aliquot 50 uL of each mix to 5 PCR tubes each

• Note: Last tube for each had less than others

• Run PCR

Step	Temp (oC)	Time
1	94	4 m
2	94	30 s
3	51	30 s
4	72	5 m
5	GOTO 2	7x
6	94	30 s
7	63	30 s
8	72	5 m
9	GOTO 6	30x
10	72	5 m
11	4	∞

VI. AGE of PCR Samples

• Make Gel:

• Measure out 0.40228 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 and pour into beaker 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 TBE buffer

• Load Gel:

• Mix samples as drops on a piece of parafilm

• Mix:

Ladder (Invtrogen 1 kb plus)	Blank	Sample
*4 uL pre-mixed	*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 ~10 uL on gel according to the following chart (except ladder which is 6 uL):

Lane 1	2	3	4	5	6	7	8	9	10	11	12	13	14
I-	I1	I2	I3	I4	I5	2-log Ladder	II-	II1	unusable	II2	II3	II4	II5

• Store DNA at 4 ^oC
• Unusual well formation in some cases

• 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 of gel

• Image Gel:

• RESULTS:

Source: Media:Srk_2012-08-09_00hr_14min.tif

Feature	Expected Size (bp)
Cassette	5152

• Arrow indicates desired product