Here, you will find protocols for the procedures we use in our lab. These have been passed on by senior members of the lab in which we work, taken from OpenWetWare.org, or adapted from reference papers by members of our team.

You may select any of these protocols to navigate directly to it on this page:

Restriction Enzyme Double Digest
Gel Extraction and Purification
Transformations
Liquid Cultures
PCR
Minipreps
Ligation
Error-Prone PCR
Nanodrop
Glycerol Stocks
PCR Purification

Materials:

• 22 uL dH2O
• 1 uL BSA
• 5 uL Buffer
• 20 uL Template
• 1 uL Enzyme 1
• 1 uL Enzyme 2

Buffer Compatibility Chart:
Number indicates percent activity of enzyme in given solution.

	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.

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.

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.

Materials

• LB
• Plated colonies of cells
• Antibiotic stock

Procedure

• Working near a flame or in a laminar flow hood, add 5 mL of LB to each falcon tube.
• Add the appropriate amount of antibiotic.
  • For example, 5 uL of 35 mg/mL chloramphenicol stock or 10 uL of 100 mg/mL carbenicillin stock.
• With a tip, scoop a colony from your agar plate and place it in the falcon tube.
• Incubate overnight at 37 °C.

Mix

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

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.

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

Mix

• 5ul 10x buffer
• 2.5ul Unbalanced DNTPs
• 1ul Forward primer
• 1ul Reverse primer
• 10uL 25 mM MgCl2
• 7.5uL 1 mM MnCl2
• 0.1ul Template
• .5ul Taq
• 22.4ul dH2O

Unbalanced DNTP Mix

• 20uL dATP
• 20uL dGTP
• 100uL dCTP
• 100uL dTTP
• 260uL H2O

(Store at -20 C)

• Log in to nanodrop program.
• Moisten a Kim wipe and clean the pedestal.
• Apply 2ul H2O to pedestal and click 'OK'.
• Press 'Blank' button.
• Wipe blank from pedestal using Kim wipe.
• Apply 2ul of desired sample to pedestal.
• Click 'measure'.
• Print results.

• Make liquid culture of desired cell strain. Grow for 12-16hrs if using Dh5a.
• Mix 800ul of grown liquid culture with 800ul of 80% glycerol. Shake with vigor.
• Flash freeze using liquid nitrogen.
• Keep at -80C
  • Note: When streaking plates from glycerol stock, it is important to leave glycerol stock tube on dry ice.

• For every volume of PCR product, add 2 volumes Binding Buffer. (ex. add 100ul Binding Buffer to 50ul PCR reaction)
• Transfer to spin column and spin for 1 minute.
• Discard flowthrough.
• Add 500ul Wash Buffer and spin for 1 minute.
• Discard flowthrough.
• Spin for 2 minutes.
• Put spin column in clean 1.5ml tube and elute with desired volume.

Competent Cells

Rehydration