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 14 degrees CelsiusNote: 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 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 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 37 degrees Celsius 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 50 degrees CelsiusAdd 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 4 degrees Celsius
Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)
300 mL DI H2O + 11 g LB agarAutoclavePut control thermometer in H2O (from the sink)Select vented container mode (Do Not Change Program)
Mix well after autoclaving; let cool to 50 degrees CelsiusAdd antibiotic (50 to 100 µg/mL) (15 mg total)Weigh on paperAdd to 0.5 mL DI H2OAdd to LB mixture when cool enough
PlateUnder flame open lids of all platesSlowly pour agar into plate, avoiding bubbles, when it touches all edges stop pouringLet sit under flames until gel solidifiesReplace 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 rpmInoculate 0.25 mL of the overnight strain into 25 mL of LBShake at 37oC until the OD650 is 0.6-0.7Harvest cells and resuspend in 12.5 mL ice cold 0.1M MgCl2Harvest immediately and resuspend in 7.5 mL cold 0.1M CaCl2Leave on ice for 30 minutes. Harvest and resuspend in 2.5 mL cold 0.1M CaCl2Leave on ice for 30 minutesFor 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 tubeAdd 250 µL Buffer P2 and mix thoroughly by inverting the tube 4-6 timesAdd 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 timesCentrifuge for 10 minutes at 13000 rpm (~17900g) in a table-top microcentrifugeApply the supernatant (from step 4) to the QIA prep spin column by decanting or pipettingCentrifuge for 30-60 seconds. Discard the flow-throughWash QIA prep spin column by adding 0.75 mL Buffer PE and centrifuging for 30-60 secondsDiscard the flow-through, and centrifuge for 1 minute to remove residual wash bufferTo 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 cellsPlace in -80oC freezer
Transformation Protocol
With a pipette tip, punch a hole through the foil cover of the DNA plateAdd 10 µL of DI waterThaw competent cells on iceAdd 1-2 µL of resuspend DNA and 50 µL of thawed competent cells to labeled tubesIncubate the cells on ice for 30 minutesHeat shock the cells at 42 degrees Celsius for 45 secIncubate the cells on ice for 2 minutesUnder flame, add 450 µL SOC brothIncubate at 37 degrees Celsius for 1 hour while rotating or shaking at 300rpmSpread cells on appropriate antibiotic LB plates (usually 100 µL)Incubate at 37 degrees Celsius for 18-24 hoursTake a colony, put in 3 mL of LB + appropriate antibioticUse resulting culture to miniprep DNA and make your own glycerol stock
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.
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