Ligation Protocol
Determine insert to vector ratios</li>Calculate the amount of insert needed if 50ng of vector is used (can use different amount of vector)</li>In a PCR tube add the following:50ng of vector</li>Amount of insert based on ratios (calculated in second step)</li>2uL of buffer</li>2uL of DNA ligase</li>Amount of water to bring total volume to 20uL</li>
</li>Incubate overnight at 14 degrees Celsius</li>Note: We used T4 DNA ligase and buffer from NEB
Excise DNA fragment from the agarose gel with a clean, sharp scalpel</li>Weigh the gel slice in a microcentrifuge tube.</li>Add 3 volumes of Buffer QG to 1 volume of gel (100mg~100uL)</li>Incubate at 50 degrees Celsius for 10 min (until the gel slice has completely dissolved)</li>After the gel slice has dissolved completely, check that the color of the mixture is yellow</li>Apply the sample to a QIAquick column, and centrifuge for 1 min</li>Maximum volume of the column is 800uL. For samples larger than this, simply load and spin again.
</li>Discard flow-through and place QIAquick column back in the same collection tube</li>To wash, add 750uL of Buffer PE to column and centrifuge for 1 min.</li>Discard the flow-through and centrifuge for additional 1 min. at 13,000rpm</li>Place QIAquick column into a clean 1.5 mL microcentrifuge tube</li>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.</li>
Gel Electrophoresis Protocol
Making a 1% agarose gel100mL 1X TBE buffer</li>1g agarose</li>microwave until agarose dissolves</li>let mixture cool</li>when cool add 8-10uL ethidium bromide</li>stir gently, let cool</li>pour into plate with comb already in place</li>let harden</li>
</li>Using the gelAdd loading buffer to DNA (for 100uL DNA, add 20uL loading buffer)</li>Load 2uL of DNA ladder into the gel</li>Load DNA into the gel</li>Run at 130V for 30min-1hr</li>
Digestion Protocol
</li>Using a microcentrifuge tube add the following:~3000-5000 ng of DNA</li>10uL Buffer 4</li>10uL BSA</li>5uL of appropriate enzyme (if doing a double digest, use 5 uL of both enzymes)</li>Amount of H2O needed to make final volume 100uL</li>
</li>Incubate at 37 degrees Celsius for 1hr and 30min</li>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
200 mL LB broth</li>AutoclavePut control thermometer in H2O (from the sink)</li>Select vented container mode (Do Not Change Program)</li>
</li>Let cool to 50 degrees Celsius</li>Add antibiotic (50-100 ug/mL) (10 mg total)Weigh on paper</li>Add to 0.5 mL DI H2O</li>Add to LB mixture when cool enough</li>
</li>Store at 4 degrees Celsius</li>
Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)
300 mL DI H2O + 11 g LB agar</li>AutoclavePut control thermometer in H2O (from the sink)</li>Select vented container mode (Do Not Change Program)</li>
</li>Mix well after autoclaving; let cool to 50 degrees Celsius</li>Add antibiotic (50 to 100 µg/mL) (15 mg total)Weigh on paper</li>Add to 0.5 mL DI H2O</li>Add to LB mixture when cool enough</li>
</li>PlateUnder flame open lids of all plates</li>Slowly pour agar into plate, avoiding bubbles, when it touches all edges stop pouring</li>Let sit under flames until gel solidifies</li>Replace lids on plates</li>
</li>Store upside down at 4 degrees Celsius</li>
Preparing Competent Cells Protocol
Place 1 colony in 5 mL of LB (with antibiotics if appropriate) Grow overnight at 37 degrees Celsius and 200-300 rpm</li>Inoculate 0.25 mL of the overnight strain into 25 mL of LB</li>Shake at 37oC until the OD650 is 0.6-0.7</li>Harvest cells and resuspend in 12.5 mL ice cold 0.1M MgCl2</li>Harvest immediately and resuspend in 7.5 mL cold 0.1M CaCl2</li>Leave on ice for 30 minutes. Harvest and resuspend in 2.5 mL cold 0.1M CaCl2</li>Leave on ice for 30 minutes</li>For long term storage, use 0.1M CaCl2 in 15% glycerol at step 6 and store cells at -800 degrees Celsius</li>
Note: Harvest cells at 5000 rpm for 10 minutes at 4 degrees Celsius
Miniprep Protocol (from QIAprep Spin Miniprep Kit)
Harvest cells at 5400g 10 minutes 40 degrees Celsius (possibly program 1)</li>Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube</li>Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4-6 times</li>Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times</li>Centrifuge for 10 minutes at 13000 rpm (~17900g) in a table-top microcentrifuge</li>Apply the supernatant (from step 4) to the QIA prep spin column by decanting or pipetting</li>Centrifuge for 30-60 seconds. Discard the flow-through</li>Wash QIA prep spin column by adding 0.75 mL Buffer PE and centrifuging for 30-60 seconds</li>Discard the flow-through, and centrifuge for 1 minute to remove residual wash buffer</li>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.
</li>
Preparing Glycerol Stock Protocol
Add 150 µL of 50% glycerol to 350 µL of cells</li>Place in -80oC freezer</li>
Transformation Protocol
With a pipette tip, punch a hole through the foil cover of the DNA plate</li>Add 10 µL of DI water</li>Thaw competent cells on ice</li>Add 1-2 µL of resuspend DNA and 50 µL of thawed competent cells to labeled tubes</li>Incubate the cells on ice for 30 minutes</li>Heat shock the cells at 42 degrees Celsius for 45 sec</li>Incubate the cells on ice for 2 minutes</li>Under flame, add 450 µL SOC broth</li>Incubate at 37 degrees Celsius for 1 hour while rotating or shaking at 300rpm</li>Spread cells on appropriate antibiotic LB plates (usually 100 µL)</li>Incubate at 37 degrees Celsius for 18-24 hours</li>Take a colony, put in 3 mL of LB + appropriate antibiotic</li>Use resulting culture to miniprep DNA and make your own glycerol stock</li>
Point mutation Protocol
1) Create reaction mixture
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.