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Team:Virginia/Protocols
From 2008.igem.org
Contents |
QIAGEN Mini-Prep
- Spin cell broth down in centrifuge at >13200 revolutions per minute in microcentrifuge (generally 4 minutes is adequate).
- Pour off remaining broth and resuspend in 250 uL of Buffer P1 (LyseBlue is nice to have).
- Add 250 uL Buffer P2 (for cell lysis). Invert microcentrifuge tubes 4-6 times. Do NOT allow to run for more than 5 minutes.
- Add 350 uL Buffer N3 and invert tubes another 4-6 times.
- Spin in microcentrifuge at >13000 revolutions per minute for ten minutes.
- Transfer supernatant to provided spin column.
- Run spin column in microcentrifuge for 60 seconds. Discard flow-through.
- Add 0.5 mL Buffer PB. Centrifuge for 60 seconds. Discard flow-through.
- Add 0.75 mL Buffer PE. Centrifuge for 60 seconds. Discard flow through.
- Centrifuge for an additional 60 seconds.
- Transfer spin-column top to a microcentrifuge tube for collection.
- Add 50 uL Buffer EB to center of column. Allow to sit for one minute.
- Centrifuge for one minute. The clear liquid in the bottom is your DNA solution.
Gel Electrophoresis (Making the gel)
- In a 100 mL Erlenmeyer flask, place and pour: 0.5 g Agar and 50 mL 1X TAE Buffer
- Microwave for 30 seconds and then swirl the flask
- Microwave for 15 seconds (caution: do not let the solution boil out of the flask)
- Pipette 2 microliters of Ethidium Bromide into the gel solution and then swirl flask
- Ethidium Bromide stains the plate for DNA to be observed under UV light
- NOTE: Ethidium Bromide is a hazardous chemical and must be disposed of properly - this includes the gel itself
- Setup gel holder with desired size and number comb perpendicular leads so that liquid gel stays contained
- Pour all of hot gel into gel holder, cover with saran wrap and leave out to solidify
Gel Electrophoresis (Loading & Running)
- Align wells on the side of the negetive(black) lead
- Fill with 1X TAE buffer to cover gel with thin layer of buffer
- carefully remove the comb
- Using small epindorf tubes mix together DNA and 6X loading dye with a 6:1 ratio (this ratio is dependant on the loading dye)
- Mix 1 uL of 1kb ladder with 5 uL of water and 1 uL of 6X loading maintaining the ratio
- Carefully pipette the DNA mixtures into the wells being careful not to pierce the bottom of the wells
3A assembly Ligation
- Assuming the 3 parts that are going to be ligated are cut appropriately
- First part cut at EcoRI and SpeI
- Second part cut at XbaI and PstI
- Back bone/Vector cut at EcoRI and PstI
- this works because S and X form a scar and the other restriction sites are preserved and the ligated insert keeps the same format with EX as a prefix and SP as a suffix
- the back bones should have a different antibiotic resistance than the 2 inserts to guarantee that any colonies that grow are products of a successful ligation
- Note: should not be done in DB3.1 cells
- For 20ul mix:
- 4 ul water
- 2 ul T4 10X ligase buffer
- 6 ul insert 1
- 6 ul insert 2
- 1 ul vector
- 1 ul T4 DNA ligase (enzyme)
- For 50ul mix:
- 17 ul water
- 5 ul T4 10X ligase buffer
- 12 ul insert 1
- 12 ul insert 2
- 2 ul vector
- 2 ul T4 DNA ligase (enzyme)
