Protocols
Ligation Protocol
Determine insert to vector ratiosCalculate 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 vectorAmount of insert based on ratios (calculated in second step)2uL of buffer2uL of DNA ligaseAmount of water to bring total volume to 20uL
Incubate overnight at 14oCNote: 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 scalpelWeigh 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 yellowApply the sample to a QIAquick column, and centrifuge for 1 minMaximum 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 tubeTo wash, add 750uL of Buffer PE to column and centrifuge for 1 min.Discard the flow-through and centrifuge for additional 1 min. at 13,000rpmPlace QIAquick column into a clean 1.5 mL microcentrifuge tubeTo 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 gel100mL 1X TBE buffer1g agarosemicrowave until agarose dissolveslet mixture coolwhen cool add 8-10uL ethidium bromidestir gently, let coolpour into plate with comb already in placelet harden
Using the gelAdd loading buffer to DNA (for 100uL DNA, add 20uL loading buffer)Load 2uL of DNA ladder into the gelLoad DNA into the gelRun at 130V for 30min-1hr
Digestion Protocol
Using a microcentrifuge tube add the following:~3000-5000 ng of DNA10uL Buffer 410uL BSA5uL 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 30minNote: 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 brothAutoclavePut control thermometer in H2O (from the sink)Select vented container mode (Do Not Change Program)
Let cool to 50oCAdd antibiotic (50-100 ug/mL) (10 mg total)Weigh on paperAdd to 0.5 mL DI H2OAdd to LB mixture when cool enough
Store at 4oC
Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)
Preparing Competent Cells Protocol
Miniprep Protocol (from QIAprep Spin Miniprep Kit)
Preparing Glycerol Stock Protocol
Transformation Protocol
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