Team:UT Dallas/Protocols

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Protocols

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 14oC

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 50oC 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 37oC 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.