Restriction Enzyme Double Digest

Restriction Enzyme Double Digest Materials:

22 uL dH2O *1 uL BSA * 5 uL Buffer * 20 uL Template * 1 uL Enzyme 1 * 1 uL Enzyme 2 Buffer Compatibility Chart {| border="1" ! !1 !2 !3 !4 |- | '''EcoRI''' || 100 || 100 || 100 || 100 |- | '''SpeI''' || 75 || 100 || 25 || 100 |- | '''PstI''' || 75 || 75 || 100 || 50 |- | '''NheI''' || 100 || 100 || 10 || 100 |- | '''XbaI''' || 0 || 100 || 75 || 100 |} ====Procedure==== * Mix reactants thoroughly. * Place at 37 C for 3 hours. * Increase to 80 C for 20 minutes to kill enzymes (some enzymes need only a 65 C heatkill, check enzyme). * Run on a gel and extract product.

Gel Extraction/Purification Procedure

Materials * GeneJET Gel Extraction Kit * Binding Buffer (1 uL for every mg of agarose gel) * 700 uL of Wash Buffer * 50 uL of Elution Buffer Procedure * Add the binding buffer to the gel slice in a microcentrifuge tube. * Incubate the gel mixture at 50-60 °C for 10 minutes (until melted). * Transfer the solution to a GeneJET purification column. * Centrifuge for 30-60 seconds at 12000 x g and discard the flow through. * Add Wash Buffer and centrifuge for 1 minute. * Discard flow through, then centrifuge empty column for 1 minute. * Transfer the column into a fresh 1.5 ml microfuge tube. * Add Elution Buffer. * Centrifuge for 1 minute and collect the flow-through.

Transformations

Materials * Competent cells * DNA template * 800 uL of LB * LB+antibiotic plates Procedure * Thaw competent cells on ice. * Transfer 50 uL of competent cells to chilled falcon tubes. * Add 1 uL of template to cells (2.5 uL if dilute). * Incubate on ice for 30 minutes. * Heat schock in 42 °C water bath for 90 seconds. * Immediately place back onto ice for 2 minutes. * Add 800 uL of LB to each tube. * Incubate at 37 °C for 1 hour. * Place 200 uL of the transformed cells on plates containing LB and the appropriate antibiotic. * Incubate overnight at 37 °C.

Liquid Cultures

Materials * LB * Plated colonies of cells * Antibiotic stock Procedure * In a sterile environment, add 4 mL of LB to each falcon tube. * Add the appropriate amount of antibiotic. ** for carb, add 8 uL. * With a tip, scoop a colony and place it in the falcon tube. * Incubate overnight at 37 °C.

PCR

Mix *10ul Q solution *5ul 10x buffer *1.25ul DNTPs *1ul Forward primer *1ul Reverse primer *1ul Template *.3ul Taq *.15ul PFU *30ul dH2O

Minipreps

Materials * Liquid culture * Miniprep kit (QIAprep Spin Miniprep Kit) Procedure * Centrifuge liquid culture of cells. * Discard the supernatant. * Resuspendd the pelleted cells in 250 uL of Buffer P1 and transfer to a microcentrifuge tube. * Add 250 uL of Buffer P2. **Invert 4-6 times until the solution become clear. * Add 350 uL Buffer N3. ** Invert 4-6 times. * Centrifuge for 10 minutes at 17900xg. * Apply the supernatant to a spin column. ** Centrifuge for 1 minute and discard the flow-through. * Wash the spin column with 0.5 ml Buffer PB. ** Centrifuge for 1 minute and discard the flow through. * Wash the spin column with 0.75 ml Buffer PE. ** Centrifuge for 1 minute and discard the flow through. * Centrifuge an additional 1 minute to remove residual wash buffer. * In a clean 1.5 ml microcentrifuge tube, elute DNA with 50 ul Buffer EB. * Centrifuge for 1 minute and collect the flow-through.

Ligations

Materials * Digested vector * Digested insert * Water * T4 DNA ligase. * T4 DNA ligase buffer. Procedure * Mix these materials in the amounts determined by the reaction volume calculator. [[media:UC_Davis_Reaction_Volume_Calculator.xls‎]]