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From 2012.igem.org

I. Agarose Gel Electrophoresis (AGE) of PCR Samples

• Make Gel:

• Measure out 0.5 g 0.50497 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 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 ~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
NEB 2-log ladder	Blank	1.1	1.2	2.1	2.2	3.1	3.2	4.1	4.2	5.1	5.2	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_2012-07-18.tif

Feature	Expected Size (bp)
Leftover pBR322	4361
Leftover pBAV1K-T5-gfp	3653
pT5 promoter	107
LacZ	3137
CAT*	729
tetr	1246
T1 terminator	94

I think that this ladder is unreliable because it puts our plasmid templates in the range of 6000-10000 bp which is incorrect. If we translate the ladder accordingly, it could be that reactions 3, 4 and 5 were successful. I will run the gel again with a 100 bp ladder to check on them.

II. Agarose Gel Electrophoresis (AGE) of PCR Samples

• Make Gel:

• Measure out 0.5 g 0.503 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 (100 bp ladder)	Blank	Sample
*Premixed from Isaacs Lab	*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
100 bp ladder	Blank	1.1	1.2	2.1	2.2	3.1	3.2	4.1	4.2	5.1	5.2

• Store DNA at 4 ^oC
• Had a lot of trouble loading the gel due to my allergies. This may impact the quality of the image.

• 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 halfway down the gel

• Image Gel:

• RESULTS:

Source: Media:Srk_2012-07-18_2.tif

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