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From 2012.igem.org

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Revision as of 23:23, 3 October 2012

I. PCR

• Mix Reagents:

• Mix according to the following table, where 2 = LacZ, 3 = CAT*, 4 = tet^r, 0.1-0.3 = +template, and 0.4 = -template

• For example, 2.1 = lacZ sample, 2.4 = lacZ control

	2.1	2.2	2.3	2.4	3.1	3.2	3.3	3.4	4.1	4.2	4.3	4.4
Reagent	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)
water	33.5	33.5	33.5	33.5	33.5	33.5	33.5	33.5	32.5	32.5	32.5	33.5
5x Phusion HiFi buffer	10	10	10	10	10	10	10	10	10	10	10	10
10 mM dNTP mix	1	1	1	1	1	1	1	1	1	1	1	1
10 uM primer (F)	2.5 (p2f_2)	2.5 (p2f_2	2.5 (p2f_2)	2.5 (p2f_2)	2.5 (p3f_2)	2.5 (p3f_2)	2.5 (p3f_2)	2.5 (p3f_2)	2.5 (p4f)	2.5 (p4f)	2.5 (p4f)	2.5 (p4f)
10 uM primer (R)	2.5 (p2r_2)	2.5 (p2r_2)	2.5 (p2r_2)	2.5 (p2r_2)	2.5 (p3r)	2.5 (p3r)	2.5 (p3r)	2.5 (p3r)	2.5 (p4r_2)	2.5 (p4r_2)	2.5 (p4r_2)	2.5 (p4r_2)
Template	ECNR2 colony*	ECNR2 colony*	ECNR2 colony*	0	ECFI5 colony*	ECFI5 colony*	ECFI5 colony*	0	1 (pBR322)	1 (pBR322)	1 (pBR322)	0
Phusion HiFi polymerase	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5

*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. Agarose Gel Electrophoresis (AGE) of PCR Samples

• Make Gel:

• Measure out 0.5 g 0.50238 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)	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 ~10 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	12	13	14	15
NEB 2-log ladder	Blank	2.1	2.2	2.3	2.4	3.1	3.2	3.3	3.4	4.1	4.2	4.3	4.4	NEB 2-log ladder

• Store DNA at 4 ^oC

• 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 near the bottom of the gel

• Image Gel:

• RESULTS:

Source: Media:Srk_2012-07-21_17hr_37min.tif

Feature	Expected Size (bp)
Leftover pBR322	4361
LacZ	3151
CAT*	729
tetr	1265