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Team:UT Dallas/Notebook
From 2013.igem.org
Protocols
Determine insert to vector ratios Calculate the amount of insert needed if 50ng of vector is used (can use different amount of vector) In a PCR tube add the following: Incubate overnight at 14 degrees Celsius Excise DNA fragment from the agarose gel with a clean, sharp scalpel Weigh the gel slice in a microcentrifuge tube. Add 3 volumes of Buffer QG to 1 volume of gel (100mg~100uL) Incubate at 50 degrees Celsius for 10 min (until the gel slice has completely dissolved) After the gel slice has dissolved completely, check that the color of the mixture is yellow Apply the sample to a QIAquick column, and centrifuge for 1 min Discard flow-through and place QIAquick column back in the same collection tube To wash, add 750uL of Buffer PE to column and centrifuge for 1 min. Discard the flow-through and centrifuge for additional 1 min. at 13,000rpm Place QIAquick column into a clean 1.5 mL microcentrifuge tube To elute DNA, add 50uL of Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min. and then centrifuge the column for 1 min.
Making a 1% agarose gel Using the gel Using a microcentrifuge tube add the following: Incubate at 37 degrees Celsius for 1hr and 30min 200 mL LB broth Autoclave Let cool to 50 degrees Celsius Add antibiotic (50-100 ug/mL) (10 mg total) Store at 4 degrees Celsius
300 mL DI H2O + 11 g LB agar Autoclave Mix well after autoclaving; let cool to 50 degrees Celsius Add antibiotic (50 to 100 µg/mL) (15 mg total) Plate Store upside down at 4 degrees Celsius
Place 1 colony in 5 mL of LB (with antibiotics if appropriate) Grow overnight at 37 degrees Celsius and 200-300 rpm Inoculate 0.25 mL of the overnight strain into 25 mL of LB Shake at 37oC until the OD650 is 0.6-0.7 Harvest cells and resuspend in 12.5 mL ice cold 0.1M MgCl2 Harvest immediately and resuspend in 7.5 mL cold 0.1M CaCl2 Leave on ice for 30 minutes. Harvest and resuspend in 2.5 mL cold 0.1M CaCl2 Leave on ice for 30 minutes For long term storage, use 0.1M CaCl2 in 15% glycerol at step 6 and store cells at -800 degrees Celsius Harvest cells at 5400g 10 minutes 40 degrees Celsius (possibly program 1) Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4-6 times Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times Centrifuge for 10 minutes at 13000 rpm (~17900g) in a table-top microcentrifuge Apply the supernatant (from step 4) to the QIA prep spin column by decanting or pipetting Centrifuge for 30-60 seconds. Discard the flow-through Wash QIA prep spin column by adding 0.75 mL Buffer PE and centrifuging for 30-60 seconds Discard the flow-through, and centrifuge for 1 minute to remove residual wash buffer To elute DNA, place the QIA prep column in a clean 1.5 mL microcentrifuge tube. Add 50 µL Buffer EB or water to the center of each QIA prep spin column, let stand for 1 minute and centrifuge for 1 minute.
Add 150 µL of 50% glycerol to 350 µL of cells Place in -80oC freezer
With a pipette tip, punch a hole through the foil cover of the DNA plate Add 10 µL of DI water Thaw competent cells on ice Add 1-2 µL of resuspend DNA and 50 µL of thawed competent cells to labeled tubes Incubate the cells on ice for 30 minutes Heat shock the cells at 42 degrees Celsius for 45 sec Incubate the cells on ice for 2 minutes Under flame, add 450 µL SOC broth Incubate at 37 degrees Celsius for 1 hour while rotating or shaking at 300rpm Spread cells on appropriate antibiotic LB plates (usually 100 µL) Incubate at 37 degrees Celsius for 18-24 hours Take a colony, put in 3 mL of LB + appropriate antibiotic Use resulting culture to miniprep DNA and make your own glycerol stock
5 ul 10x buffer
10-100 ng DNA
1 ul of foward primer
1 ul of reverse primer
1 ul of dNTP'S
1.5 ul of Quik Solution reagent
Bring to 50 ul with NF-H20
*Then add 1ul Quik Change Lightning Enzyme
2)Run thermo-cycler (program--mutate)
1 cycle: @ 95C 2 minutes
18 cycles:
a) 95C x 20 seconds
b) 60C x 10 seconds
c) 68C x 30 seconds/kb per plasmid length
1 cycle: 68C 5 minutes
3) Then add 2 ul of DpnI enzyme directly to each amplification reaction
4) Pipette up & down several times
5) Incubate @ 37C x 5 minutes to digest the parent DNA (cuts methylated dna)
6)Then transform.
Ligation Protocol
50ng of vector Amount of insert based on ratios (calculated in second step) 2uL of buffer 2uL of DNA ligase Amount of water to bring total volume to 20uL
Note: We used T4 DNA ligase and buffer from NEB
Gel Purification Protocol (QIAquick Gel Extraction Kit)
Maximum volume of the column is 800uL. For samples larger than this, simply load and spin again.
Gel Electrophoresis Protocol
100mL 1X TBE buffer 1g agarose microwave until agarose dissolves let mixture cool when cool add 8-10uL ethidium bromide stir gently, let cool pour into plate with comb already in place let harden
Add loading buffer to DNA (for 100uL DNA, add 20uL loading buffer) Load 2uL of DNA ladder into the gel Load DNA into the gel Run at 130V for 30min-1hr
Digestion Protocol
~3000-5000 ng of DNA 10uL Buffer 4 10uL BSA 5uL of appropriate enzyme (if doing a double digest, use 5 uL of both enzymes) Amount of H2O needed to make final volume 100uL
Note: We used the following enzymes from NEB: EcoRI-HF, PstI-HF, SpeI, and XbaI. All of which can be double digested with each other using Buffer 4.
Preparing LB+Appropriate Antibiotic Protocol
Put control thermometer in H2O (from the sink) Select vented container mode (Do Not Change Program)
Weigh on paper Add to 0.5 mL DI H2O Add to LB mixture when cool enough
Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)
Put control thermometer in H2O (from the sink) Select vented container mode (Do Not Change Program)
Weigh on paper Add to 0.5 mL DI H2O Add to LB mixture when cool enough
Under flame open lids of all plates Slowly pour agar into plate, avoiding bubbles, when it touches all edges stop pouring Let sit under flames until gel solidifies Replace lids on plates
Preparing Competent Cells Protocol
Note: Harvest cells at 5000 rpm for 10 minutes at 4 degrees Celsius
Miniprep Protocol (from QIAprep Spin Miniprep Kit)
Preparing Glycerol Stock Protocol
Transformation Protocol
Point mutation Protocol
1) Create reaction mixture5 ul 10x buffer
10-100 ng DNA
1 ul of foward primer
1 ul of reverse primer
1 ul of dNTP'S
1.5 ul of Quik Solution reagent
Bring to 50 ul with NF-H20
*Then add 1ul Quik Change Lightning Enzyme
2)Run thermo-cycler (program--mutate)
1 cycle: @ 95C 2 minutes
18 cycles:
a) 95C x 20 seconds
b) 60C x 10 seconds
c) 68C x 30 seconds/kb per plasmid length
1 cycle: 68C 5 minutes
3) Then add 2 ul of DpnI enzyme directly to each amplification reaction
4) Pipette up & down several times
5) Incubate @ 37C x 5 minutes to digest the parent DNA (cuts methylated dna)
6)Then transform.
