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From 2012.igem.org

Contents 1 I. Check Overnight Cultures for Growth 2 II. AGE of PCR Samples 3 III. Make 15% Glycerol Stocks (6 total) from Overnight Cultures 4 IV. Miniprep (QIAGEN) PCR Cultures

I. Check Overnight Cultures for Growth

• RESULTS: All cultures showed growth.

II. AGE of PCR Samples

• Make Gel:

• Measure out 0.4 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 in refrigerator, 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)	Sample
*5 uL pre-mixed	*10 uL DNA *2 uL 6X Loading Dye

• Load ~12 uL on gel according to the following chart:

Lane 1	2	3	4	5	6	7
NEB 2-log ladder	- control	1:2 #1	1:1 #1	1:1 #2	1:1 #3	1:1 #4

• 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 down the gel

• Image Gel:

• RESULTS:

Source: Media:Srk_2012-09-06_14hr_55min.tif

Feature	Expected Size (bp)
Cassette	5152

III. Make 15% Glycerol Stocks (6 total) from Overnight Cultures

• Transfer 600 uL of each colony PCR liquid culture to a microcentrifuge tube (x1 each)
• Add 200 uL of 60% glycerol to each tube (x1 each)
• Vortex to mix thoroughly
• Store tubes in the -80 ^oC freezer

IV. Miniprep (QIAGEN) PCR Cultures

• Pellet cells in microcentrifuge tube in tabletop microcentrifuge at 13000 rpm for 3 min
• Resuspend pelleted bacterial cells in 250 uL Buffer P1 (from fridge, shake before opening) in each microcentrifuge tube by vortexing/pipetting
• Add 250 uL Buffer P2 and mix gently by inverting the tubes 6 times until solutions were homogeneous and blue (they were only a very light blue)
• Add 350 uL Buffer N3 and mix immediately and thoroughly by inverting the tubes 10-15 times until suspension is colorless and cloudy
• Centrifuge for 10 min at 13000 rpm in tabletop microcentrifuge

• Note: Pellets were compact but spread across upper wall of tubes.

• Apply the supernatants to the QIAprep spin column by pipetting
• Centrifuge for 1 min, discard flow-through
• Wash QIAprep spin column by adding 0.5 mL Buffer PB and centrifuging for 1 min, discard flow-through
• Wash QIAprep spin column by sdding 0.75 mL Buffer PE and centrifuging for 1 min, discard flow-through
• Centrifuge for 1 min to remove residual wash buffer, prepare microcentrifuge tubes
• Place the QIAprep columns in clean microcentrifuge tubes
• Add 50 uL Buffer EB to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min
• Save columns in separate microcentrifuge tubes in case we need to elute again