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Team:UT Dallas/protocols new
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<div id="right"> <h2><span></span>Ligation Protocol</h2> | <div id="right"> <h2><span></span>Ligation Protocol</h2> | ||
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<p></p><li type="disk">With a pipette tip, punch a hole through the foil cover of the DNA plate</li><li type="disk">Add 10 µL of DI water</li><li type="disk">Thaw competent cells on ice</li><li type="disk">Add 1-2 µL of resuspend DNA and 50 µL of thawed competent cells to labeled tubes</li><li type="disk">Incubate the cells on ice for 30 minutes</li><li type="disk">Heat shock the cells at 42 degrees Celsius for 45 sec</li><li type="disk">Incubate the cells on ice for 2 minutes</li><li type="disk">Under flame, add 450 µL SOC broth</li><li type="disk">Incubate at 37 degrees Celsius for 1 hour while rotating or shaking at 300rpm</li><li type="disk">Spread cells on appropriate antibiotic LB plates (usually 100 µL)</li><li type="disk">Incubate at 37 degrees Celsius for 18-24 hours</li><li type="disk">Take a colony, put in 3 mL of LB + appropriate antibiotic</li><li type="disk">Use resulting culture to miniprep DNA and make your own glycerol stock<p></p></li> | <p></p><li type="disk">With a pipette tip, punch a hole through the foil cover of the DNA plate</li><li type="disk">Add 10 µL of DI water</li><li type="disk">Thaw competent cells on ice</li><li type="disk">Add 1-2 µL of resuspend DNA and 50 µL of thawed competent cells to labeled tubes</li><li type="disk">Incubate the cells on ice for 30 minutes</li><li type="disk">Heat shock the cells at 42 degrees Celsius for 45 sec</li><li type="disk">Incubate the cells on ice for 2 minutes</li><li type="disk">Under flame, add 450 µL SOC broth</li><li type="disk">Incubate at 37 degrees Celsius for 1 hour while rotating or shaking at 300rpm</li><li type="disk">Spread cells on appropriate antibiotic LB plates (usually 100 µL)</li><li type="disk">Incubate at 37 degrees Celsius for 18-24 hours</li><li type="disk">Take a colony, put in 3 mL of LB + appropriate antibiotic</li><li type="disk">Use resulting culture to miniprep DNA and make your own glycerol stock<p></p></li> | ||
| + | |||
| + | <h2><span></span>Point mutation Protocol</h2> | ||
| + | <div class="clr"></div> | ||
| + | 1) Create reaction mixture | ||
| + | <br> 5 ul 10x buffer | ||
| + | <br> 10-100 ng DNA | ||
| + | <br> 1 ul of foward primer | ||
| + | <br> 1 ul of reverse primer | ||
| + | <br> 1 ul of dNTP'S | ||
| + | <br> 1.5 ul of Quik Solution reagent | ||
| + | <br> Bring to 50 ul with NF-H20 | ||
| + | <br> *Then add 1ul Quik Change Lightning Enzyme | ||
| + | <br>2)Run thermo-cycler (program--mutate) | ||
| + | <br> 1 cycle: @ 95C 2 minutes | ||
| + | <br> 18 cycles: | ||
| + | <br> a) 95C x 20 seconds | ||
| + | <br> b) 60C x 10 seconds | ||
| + | <br> c) 68C x 30 seconds/kb per plasmid length | ||
| + | <br> 1 cycle: 68C 5 minutes | ||
| + | <br>3) Then add 2 ul of DpnI enzyme directly to each amplification reaction | ||
| + | <br>4) Pipette up & down several times | ||
| + | <br>5) Incubate @ 37C x 5 minutes to digest the parent DNA (cuts methylated dna) | ||
| + | <br>6)Then transform. | ||
| + | <br><br> | ||
<div class="clr"> | <div class="clr"> | ||
</div> | </div> | ||
Latest revision as of 03:41, 29 September 2011
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.
