Team:UT Dallas/protocols new

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Ligation Protocol

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:

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

Incubate overnight at 14 degrees Celsius

Note: We used T4 DNA ligase and buffer from NEB

Gel Purification Protocol (QIAquick Gel Extraction Kit)

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

Maximum volume of the column is 800uL. For samples larger than this, simply load and spin again.

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.

Gel Electrophoresis Protocol

Making a 1% agarose gel

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

Using the gel

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

Using a microcentrifuge tube add the following:

~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

Incubate at 37 degrees Celsius for 1hr and 30min

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

Autoclave

Put control thermometer in H2O (from the sink)
Select vented container mode (Do Not Change Program)

Let cool to 50 degrees Celsius

Add antibiotic (50-100 ug/mL) (10 mg total)

Weigh on paper
Add to 0.5 mL DI H2O
Add to LB mixture when cool enough

Store at 4 degrees Celsius

Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)

300 mL DI H2O + 11 g LB agar

Autoclave

Put control thermometer in H2O (from the sink)
Select vented container mode (Do Not Change Program)

Mix well after autoclaving; let cool to 50 degrees Celsius

Add antibiotic (50 to 100 µg/mL) (15 mg total)

Weigh on paper
Add to 0.5 mL DI H2O
Add to LB mixture when cool enough

Plate

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

Store upside down at 4 degrees Celsius

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

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

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)

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.

Preparing Glycerol Stock Protocol

Add 150 µL of 50% glycerol to 350 µL of cells

Place in -80oC freezer

Transformation Protocol

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

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