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From 2012.igem.org

Contents 1 I. AGE of PCR Samples 2 II. AGE of PCR Samples (2) 3 III. PCR for LacZ 4 IV. AGE of PCR Samples (3)

I. AGE of PCR Samples

• Make Gel:

• Measure out 0.50131 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 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)	Sample
*5 uL DNA ladder *1 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
NEB 2-log ladder	-	P1	P2	HF1	HF2	LR1	LR2

• Store DNA at 4 ^oC
• P = Phusion, HF = KAPA HiFi, LR = KAPA LongRange

• 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 ~3/4 of gel

• Image Gel:

• RESULTS:

Source: Media:Srk_2012-08-06_13hr_02min.tif

Feature	Expected Size (bp)
LacZ	3151

II. AGE of PCR Samples (2)

Note: Repeated with more loaded per lane

• Make Gel:

• Measure out 0.50180 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 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)	Sample
*5 uL DNA ladder *1 uL 6x Loading Dye	*2 uL 6x Loading Dye *10 uL DNA

• Load ~12 uL on gel according to the following chart (except ladder which is 6 uL):

Lane 1	2	3	4	5	6	7	8
NEB 2-log ladder	-	P1	P2	HF1	HF2	LR1	LR2

• Store DNA at 4 ^oC
• P = Phusion, HF = KAPA HiFi, LR = KAPA LongRange

• 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 ~3/4 of gel

• Image Gel:

• RESULTS:

• Same as above.

III. PCR for LacZ

• Mix Reagents:

• Mix according to the following table

	1 Reaction	Phusion Master Mix (x2)	KAPA HiFi Master Mix (x2)	KAPA LongRange Master Mix (x2)
Reagent	(uL)	(uL)	(uL)	(uL)
water	33	66	66	59
5x Appropriate Buffer	10	20	20	20
10 mM dNTP mix	1.5	3	3	3
Mg2+	-	-	-	7
10 uM primer (F)	2.5 (p2f_2)	5 (p2f_2)	5 (p2f_2)	5 (p2f_2)
10 uM primer (R)	2.5 (p2r_2)	5 (p2r_2)	5 (p2r_2)	5 (p2r_2)
Template Mix	colony	-	-	-
Appropriate polymerase	0.5	1	1	1

• Touch a sterile pipette tip to one colony each for samples and smear it on the bottom of a tube.
• Transfer 50 uL of each Master Mix to 6 total tubes
• Create a separate blank under Phusion conditions

• Run PCR

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

IV. AGE of PCR Samples (3)

• Make Gel:

• Measure out 0.50114 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 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)	Sample
*5 uL DNA ladder *1 uL 6x Loading Dye	*2 uL 6x Loading Dye *10 uL DNA

• Load ~12 uL on gel according to the following chart (except ladder which is 6 uL):

Lane 1	2	3	4	5	6	7	8
NEB 2-log ladder	-	P1	P2	HF1	HF2	LR1	LR2

• Store DNA at 4 ^oC
• P = Phusion, HF = KAPA HiFi, LR = KAPA LongRange

• 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 ~3/4 of gel

• Image Gel:

• RESULTS:

Source: Media:Srk_2012-08-06_21hr_49min.tif

Feature	Expected Size (bp)
LacZ	3151