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From 2012.igem.org

Contents 1 I. Check Cultures for Growth 2 II. Miniprep (QIAGEN) Plasmid Cultures 3 III. Digest Samples of Plasmids with XhoI 4 IV. Agarose Gel Electrophoresis (AGE) of Plasmid Samples

I. Check Cultures for Growth

RESULTS (~09:30):

• Overnight cultures of plasmid strain pBAV1K-T5-gfp both grew robustly overnight. I will not centrifuge them together but do two separate Minipreps

II. Miniprep (QIAGEN) Plasmid Cultures

• (~10:00) Pellet cells in microcentrifuge tube in tabletop microcentrifuge at 13000 rpm for 3 min

• For each culture, transfer ~1 mL of cells to microcentrifuge tube, spin, discard supernatant
• Repeat until all culture has been pelleted (5 times each) and then remove residual supernatant with a pipette

• 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
• 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

III. Digest Samples of Plasmids with XhoI

• For each of the samples mix in a small tube:

• 6.4 uL dH₂O
• 1.0 uL 10x NEB Buffer 3
• 0.1 uL 100x BSA
• 2.0 uL Miniprepped DNA
• 0.5 uL XhoI enzyme

• Sample List:

Sample Number	Sample
1	pIM1463
2	pBAV1K-T5-gfp from 14 Jul 12
3	pBAV1K-T5-gfp from 15 Jul 12 (I)
4	pBAV1K-T5-gfp from 15 Jul 12 (II)

• Incubate reaction in 37 ^oC water bath for 1 hour

• NOTE: Water bath spiked to ~40 ^oC about 20 min in to reaction. Temperature was fixed and the reaction was incubated for a full hour.

• Remove tubes and put them on ice until loading gel

IV. Agarose Gel Electrophoresis (AGE) of Plasmid Samples

• Make Gel:

• Measure out 0.5 g 0.50176 g of agarose (from 2 different sources because the first ran out) 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:

• For all samples other than the digestions, mix samples as drops on a piece of parafilm
• For digestion samples, mix directly in the tube

• Mix:

Ladder (Invitrogen 1 kb plus)	Blank	Undigested (U)	Digested (D)
*5 uL DNA ladder *5 uL 6x Loading Dye	*12.5 uL dH2O *2.5 uL 6x Loading Dye	*10.5 uL dH2O *2.5 uL 6x Loading Dye *2.0 uL DNA	*2.5 uL dH2O *2.5 uL 6x Loading Dye *10 uL reaction mixture

• Load ~15 uL on gel according to the following chart:

Lane 1	2	3	4	5	6	7	8	9	10	11	12
1 kb ladder	Blank	(empty)	U1	D1	U2	D2	U3	D3	U4	D4	1 kb ladder

• Store DNA at -20 ^oC

• Run Gel:

• Close gel box and turn on power pack
• NOTE: By mistake, I used the constant mA setting at 120 mA for ~1 min before realizing that this gave a voltage that was much too high (~340 V). I stopped the gel and proceeded as listed below.
• Run gel at 50 V for 20 min to stack gel
• Run gel at 75 V until the markers have reached the bottom of the gel

• NOTE: It appears that the ladders are running slower than the other lanes.

• Image Gel:

• RESULTS:

Source: Media:Srk_2012-07-15_17hr_21min.tif