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From 2012.igem.org

I. AGE of PCR Samples

• Make Gel:

• Measure out 0.45035 g of agarose and add to a 250 mL E-flask
• Add 50 mL of 0.5x TBE buffer and swirl to mix (0.9% agarose 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
*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 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	Blank	-	+ 1	+ 2	+ 3	+ 4	+ 5

• 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 ~3/4 of gel

• Image Gel:

• RESULTS:

Source: Media:Srk_2012-08-04_14hr_27min.tif

Feature	Expected Size (bp)
Cassette	5152

II. Check ECNR2 Plate

• Growth successful for single colonies

III. PCR for LacZ

• Mix Reagents:

• Mix according to the following table

	1 Reaction	Master Mix (x6)
Reagent	(uL)	(uL)
water	33	198
5x KAPALongRange buffer	10	60
10 mM dNTP mix	1.5	9
10 uM primer (F)	2.5 (p2f_2)	15 (p2f_2)
10 uM primer (R)	2.5 (p2r_2)	15 (p2r_2)
Template Mix	colony	-
KAPA LongRange polymerase	0.5	3

• Touch a sterile toothpick to one colony each for samples 2.1-2.5 and smear it on the bottom of a tube. Do not do this for one more tube.
• Transfer 50 uL of Master Mix to each tube

• Run PCR O/N

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