Team:UC Davis/Protocols

From 2011.igem.org

Our Sponsors

Start a Family

Got a favorite BioBrick? Check our our process for expanding basic parts into part families.

Criteria

View our judging criteria for iGEM 2011 here.

Protocols

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.

All protocols can be printed by clicking the link at the top right of their description.

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
Registry Part Distribution Rehydration
Making Competent Cells

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:
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.

Gel Extraction and Purification

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

  • 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.

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.

Ligation

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

Error-Prone PCR

This PCR protocol generally introduces between one and seven mutations (i.e. single base substitutions) per 1000 bases of template DNA. This is due to an increase in the magnesium chloride concentration, the addition of manganese chloride, the low template concentration, an unbalanced solution of dNTPS (that is, A,G,C, and T are not all present at the same concentration) and the use of Taq polyermase without an error-checking enzyme.

Prepare Unbalanced DNTP Mix:

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

(Store at -20 C)

Create a Mutant PCR Mix using:

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

These values can be multiplied by 5x, 10x or however much you like, so long as the mixture is stored at -20C. Aliquot 49.4 uL of this solution into a PCR tube per each reaction and add:

  • 0.1ul Template
  • .5ul Taq

Mix the reaction thoroughly and place it in a thermal cycler. You can use the same program that you would for a conventional PCR reaction, with run times appropriate for the length of your template. We used around twenty cycles and an elongation time of 30 seconds for our promoter mutants, which took only a few hours to complete per cycle.

To increase the mutation rate, dilute the results of one error-prone PCR reaction by roughly 100x and repeat the process, using the new product as template. This will increase the number of mutations per 1000 base pairs accordingly -- after three rounds, our LacI promoter mutants showed between 1 and 7 mutations per 300 bases.

Nanodrop

  • 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.

Glycerol Stock

  • 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.

PCR Purification

  • 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.

Registry Part Distribution Rehydration

Procedure

  • Add 20ul sterile H2O to desired well in distribution plate.
  • Incubate at room temperature for ~10 minutes.
  • Transfer resuspension to microcentrifuge tube.


Making Competent Cells

Materials

  • 0.5M PIPES buffer (piperazine-1,2-bis[2-ethanesulfonic acid])(pH 6.7)
    • Dissolve 15.1g of PIPES in 80ml of pure H2O. Adjust to pH 6.7 with 5M KOH and bring volume to 100ml with pure H2O. Filter sterilize and store at -20C
  • Inoue transformation buffer
    • Dissolve 10.88g McCl2.4H2O, 2.20g CaCl2.2H2O, 18.65g KCl, and 20ml PIPES in 800ml pure H2O and fill to 1L
    • Filter sterilize and store at -20C
  • DMSO

Procedure

  • Starting around noon, pick a single bacterial colony (2-3mm in diameter) from a plate that has incubated for 16-20hrs at 37C. Transfer colony into 25ml of LB medium in a 250ml flask. Incubate for 6-8hrs at 37C with vigorous shaking.
  • At about 6:00pm, use the starter culture to inoculate three 1L flasks each containing 250ml LB. The first flask receives 6ml of starter culture, the second receives 4ml, and the third receives 2ml. Incubate all three flasks at 18-22C overnight with moderate shaking.
  • The following morning, read the OD600 of all three cultures monitoring every 45 minutes.
  • When one of the cultures reaches an OD600 of 0.55, transfer the culture vessel to an ice-water bath for 10 minutes. Discard the other cultures unless you desire to do multiple batches at once.
  • Harvest the cells by centrifugation at 2500g (3900rpm in a Sorvall GSA rotor) for 10 minutes at 4C.
  • Pour off the medium and store the open centrifuge bottle on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to walls of the centrifuge bottle or trapped in its neck.
  • Resuspend the cells gently in 80ml of ice-cold Inoue transformation buffer.
  • Harvest the cells by centrifugation at 2500g for 10 minutes at 4C
  • Pour off the medium and store the open centrifuge tube on a stack of paper towels for 2 minutes. Again, use the vacuum aspirator to remove any drops.
  • Resuspend the cells gently in 20ml of ice-cold Inoue transformation buffer.
  • Add 1.5ml of DMSO. Mix the bacterial suspension by swirling and then store it in ice for 10 minutes.
  • Working quickly, dispense aliquots of the suspensions into chilled, sterile microcentrifuge tubes. Immediately snap-freeze the competent cells by immersing the tightly closed tubes in a bath of liquid nitrogen. Store the tubes at -80C until needed for transformation.

Retrieved from "https://2011.igem.org/Team:UC_Davis/Protocols"