User:Mbennie/Notebook/Protocols

Phosphorylation

• Purpose: To add phosphate to the end of an oligio
• Use: 8 ul water, 1 ul primer, 1 ul T4 PNK 10x Buffer. Then add .5 ul of T4 PNK. Thermocycler protocol on website (Austin: PNK).

Binding

• Purpose: To allow two complementary, single-stranded DNA fragments to bond together to form a double strand piece of DNA.
• Use: Put fragments into the same tube. Run in thermocycler under Austin: Cool.

Digest

• Purpose: To cut fragment out of vector to make room for new fragment
• Use: Add appropriate buffer (10x), BSA (100x if needed), DNA (~1ug per 40 ul), 2 enzymes (Bbs1 [2 ul] and Ear1 [1 ul] per 40 ul), and the rest with water. Run under Austin: Cut and Cool.

Ligation

• Purpose: Put DNA fragment into a vector
• Use: Put 1 ul of oligio mix, 1 ul of 10x ligase buffer, 6 ul of water, and 2 ul of vector into a tube. Then add .5 ul ligase from -20. Vortex and run in thermocycler under Austin: Ligation.

Transformation/Transfection

• Purpose: Introduces vector into bacteria (transformation) or animal cell (transfection)
• Use: Thaw cells (tubes are on third shelf of -80, next middle, plates are on fourth shelf to the right). Add 2 ul of DNA to cells and incubate (in ice) for 30 minutes. Heat shock for 45 seconds. Add 300 ul liquid SOC to cells and incubate for an hour.

Solid Culture / Plate

• Purpose: To grow colonies (clusters of bacteria that are derived from and share the same piece of DNA).
• Use: Take transformed DNA and pipette onto the surface of the plate. Spread with sterile wand. Place plate upside down in the incubator overnight.

Liquid Culture

• Purpose: To grow a lot of DNA
• Use: Add 8ml of liquid LB Lennox Broth (with KAN) to a tube with 50 ul of DNA. Incubate overnight on a roller wheel.

Miniprep

• Purpose: A quick method to obtain a small amount of DNA in order to test effectiveness of insertion
• Use (Filtering): Store 1.6 ml in glycerol and put in the -80. Spin down liquid culture in centrifuge. Dump supernatant into 10% bleach solution and then follow protocol on the wall in lab.

Maxiprep

• Purpose: Make a LOT of DNA.
• Use: 250 ml solid culture overnight in 1L flask. Follow maxiprep protocol (under sink in lab near DNA synthesizer).

Changing a rotor

• Use: Replace the rotor, spin clockwise, and hold start until the new rotor is recognized.

Gel Purification

• Purpose: To extract useful DNA from a gel.
• Use: UV lightbox (low setting) is used to find desired bands. Cut these out (getting as little gel as possible) and put them in 1.5ml tubes. Determine weight of gel and then follow QAIEXII protocol in the third draw.

Colony PCR

• Purpose: To extract DNA from cell
• Use: Fill strip with 50 ul of water per well. Use pipette tip to remove colony, swirl in water, and then streak new plate (with grid sticker). Make sure there is a neg control. Incubate new plate for hour (this is back up if PCR has good results). 10 ul PCR mix, .5 ul DNA, .2 ul of each primer. Thermocycler protocol on website.

Spec

• Purpose: to determine the quantity and quality of DNA present
• Use: Use Nanodot machine. Rinse sensor with water and wipe. Blank machine with 2 ul of water or buffer. Place 2 ul of sample on sensor and measure. Clean sense with water when finished.

Preparing a Gel

• Purpose: To later run a gel (determine length of DNA fragment)
• Use: Get clean bottle and add 50 ul (small gel) or 100 ul (large gel) of 1x TAE and .5 g or 1 g of gel power (for 1% gel). Heat until single solution. Add 10 ul of sybersafe and mix. Pour into gel plate (comb already inserted). Clean bottle with water and place on dirty cart.

Running a Gel

• Purpose: To determine the length of a DNA fragment
• Use: Put gel in gel box, fill with TAE buffer, and remove comb. Add 2 ul of loading dye to samples (10 ul PCR product) and fill wells. Add 12 ul of ladder to a well. Turn on electricity and set timer.

Sequencing

• Purpose: To determine sequence of DNA
• Use: Go to sequencing web page. Use Austin’s login and enter information. Use standard priority and enter number of rxns. Enter other information (tube #, product length, primer, plasmid [vector name = pSB2K4], etc). Print out order. Determine how much DNA is required for type. Use strip, make sure each sample has 8 ul water, .1-.2 ul primer (only one), and # ul DNA. Take strip and paper to E17, 4th floor. Put paper in folder on fridge and put strip in tube. Label tube with Che and order number.