Team:Newcastle/Project/Labwork/PhilsProtocols

=Professor Phil Aldridge's Lab Protocols=

Preparing Competent E.coli cells for Heat Shock
In general Pre-culture Culture Harvest cells Freezing
 * This protocol is designed for 200 ml of cell culture which will result in 10-15 aliquots of 100 µl Ca2+-competent cells
 * Solutions required:
 * 200 ml LB media (sterile)
 * 150 ml pre-chilled 100 mM CaCl2 (sterile)
 * Incubate plate overnight
 * Pick a single colony and inoculate 5 ml LB
 * Incubate overnight at 37°C
 * Inoculate 200 ml LB with 1ml of preculture
 * Incubate culture at 37°C on a shaker with 180 - 200 rpm
 * After 2 hours start measuring the OD600
 * When the culture reaches an OD600 between 0.1 and 0.2
 * Carry out all steps on ice!!!
 * Transfer into one large centrifugation vial
 * Centrifuge cells 10 minutes at 4°C in a at 8'000 rpm
 * Carefully discard supernatant
 * Resuspend each pellet in 40 ml pre-chilled 100 mM CaCl2
 * Keep cells on ice for 40 minutes EXACTLY!!!
 * Centrifuge cells 10 minutes at 4°C at 8'000 rpm
 * Carefully discard supernatant
 * Resuspend each pellet in 1 ml pre-chilled 100 mM CaCl2
 * To the the cell concentrate add 100% glycerol to give a final concentration of 0.1%
 * Aliquot 100 ul protions of the mix into sterile microfuge tubes
 * Shock-freeze cells in liquid nitrogen
 * Store samples at -80°C

Transforming DNA "Phil Style"

 * 1) Switch on heat block in flow to “LOW”.
 * 2) Go get cells out of -80˚C and leave on ice for 30 minutes.
 * 3) Check to see if heat block is at approx 42-45˚C.
 * 4) Add DNA 1-20 ul to cells after vortexing them.
 * 5) Leave on ice for EXACTLY 30 mins.
 * 6) Place tubes in heat block for EXACTLY 50 secs.
 * 7) Transfer back to ice for 2 mins.
 * 8) After 2 mins add 0.9 ml LB and incubate for 45-60 mins. At 37˚C.
 * 9) Plate out 200 ul and 200ul of a 1:10 dilution and start praying.

Preparation of cells
 * 1) Dilute a ON culture 1:200 into at least 200 ml LB
 * 2) Grow to an OD600 between 0.1 and 0.2 (usually 2-3hrs)
 * 3) Spin down cells
 * 4) Resuspend in 40ml ice cold 0.1 M CaCl2 and leave on ice for 30 min.
 * 5) Spin down cells and resuspend in 1 ml 0.1M CaCl2
 * 6) Transfer cells to an 1.7ml tube and add 105 ul glycerol and make sure you get a homogeneous solution
 * 7) Aliquot in 100 ul volumes into clean 1.7 ml tubes and shock freeze in liquid nitrogen
 * 8) store at –80˚C until used up

Restriction Digests
In general


 * Solutions required:
 * ddH2O (sterile and filtered)
 * Restriction enzyme and buffer
 * Your DNA

Simple Test Digests

   Cloning Experiments  
 * For simple test digests the final volume should be 20 ul.
 * If you are testing vector DNA, quantify using a UV spec and dilute an amount down (usually 200 ul) to 0.05 ug/ul and use this as your working solution.
 * All digests must contain the following:
 * Sterile H2O giving a final volume of 20 ul (usually 7.5 ul)
 * 2 ul 10x restriction buffer
 * 10 ul of your DNA
 * 0.5 ul Restriction Enzyme
 * All these should be added in the above order to prevent contamination.
 * Incubate at the appropriate temperature for 1 hour then run entire sample on a 0.8% agarose gel.
 * For digests during a cloning experiment all final volumes should be 50 ul.
 * Sterile H2O giving a final volume of 50 ul
 * 5 ul 10x restriction buffer
 * your DNA
 * 1 to 2 ul Restriction Enzyme
 * For your vector DNA digest 20 ul of your working 0.05 ug/ul solution.
 * If your insert is a PCR reaction digest the lot after cleaning it up or for a subcloning digest 1-2 ug of plasmid DNA.
 * Incubate all reactions at the appropriate temperature for 3 hours.
 * For inserts (whether PCR products or fragments) run entire reaction on a gel in 2 lanes and extract DNA using Sigma Gel Extraction Kit.
 * For vectors use a standard ethanol precipitation to get rid of buffer and resuspend pellet in 20 ul ddH20.
 * Use DNA immediately for a Ligation (different protocol sheet).

Ligation Reactions
In general  Ligations   Note: INSERT DNA Page 1.67 (sambrook)
 * Before attempting a ligation you should read the information on the back
 * Ref: Molecular cloning vol 1 Sambrook et al.
 * Solutions required:
 * Digested DNA
 * T4 DNA ligase and buffer
 * All ligations should have a final volume of 20 ul.
 * Your vector DNA should have a concentration of 0.05 ug/ul at the end of all manipulations.
 * N.B. Assume maximum recovery and use your starting concentration!
 * All ligations must contain the following:
 * Sterile H2O giving a final volume of 20 ul
 * 2 ul 10x ligation buffer
 * 1 - 16 ul insert (see note)
 * 1 ul 0.05 ug/ul vector DNA
 * 1 ul T4 DNA ligase
 * Controls are very important here, especially cut vector with ligase and cut vector without ligase.
 * Incubate on the top shelf of a fridge overnight before transforming 10 - 20 ul into appropriate cells.
 * The general rule is to have excess molar amounts of insert to vector.
 * If you cut your insert out of a gel and it is SMALLER than your vector you can take 16 ul of the elution without doing any quantification.
 * This also stands for PCR products (as they are almost always smaller than the vector). However, sometimes it is not necessary to elute from a gel.

''...proportion of transformed bacterial colonies that carry recombinant plasmids. In this case, it is advisable to consider taking steps to reduce the background of colonies carrying nonrecombinant plasmids either by treating the linearized plasmid DNA with phosphatase or by adopting another cloning strategy so that the recombinant plasmid can be constructed by directional cloning.''  Textbook: Molecular Cloning: A Laboratory Manual by Joseph Sambrook, David W Russell

Ethanol Precipitating
Preparation of DNA
 * To a 50 ul solution of DNA add 5 ul 3M NaAc pH 5.2 and 140 ul 100% Ethanol (i.e. 0.1 Vol NaAc 2.5 Vols Ethanol).
 * Precipitate for 30 min (at RT or -80°C) before spinning at full speed for 15 minutes.
 * Wash the pellet with 500 ul 70 % Ethanol and spin for a further 10 mins.
 * Dry pellet in the speed vac for 3 to 5 mins.
 * Resuspend the pellet in required vol of required buffer.

Alkaline Dephosphorylation
In general Preparation of DNA Heat Inactivation and Preparation for ligation
 * Solutions required:
 * ddH2O (sterile and filtered)
 * Alkaline Phosphatase and buffer
 * Your DNA
 * After a 3 hr digest of your DNA add 5 ul 3M NaAc pH 5.2 and 140 ul 100% Ethanol
 * Precipitate for 30 min before spinning at full speed for 15 minutes
 * Wash the pellet with 500 ul 70 % Ethanol and spin for a further 10 mins.
 * Dry pellet in the speed vac for 3 to 5 mins.
 * Resuspend the pellet in 50 ul H2O
 * Aliquot 8 ul into a clean tube and keep this is your phosphorylated control
 * Add 5 ul AP buffer and 3 ul Alkaline Phosphatase to the remaining 42 ul
 * Incubate at 37 °C for 1 to 3 hrs
 * Add the following to your dephosporylation reaction
 * 10 ul 10x TNE
 * 5 ul 10 % SDS
 * 35 ul H2O


 * Incubate at 68°C for 15 min to inactivate the enzyme
 * Add 10 ul 3M NaAc pH 5.2 and 275 ul 100% Ethanol
 * Precipitate for 30 min before spinning at full speed for 15 minutes
 * Wash the pellet with 500 ul 70 % Ethanol and spin for a further 10 mins.
 * Dry pellet in the speed vac for 3 to 5 mins.
 * Resuspend the pellet in 20 ul H2O


 * Use DNA immediately for a Ligation (different protocol sheet).

Phil’s mini method for Alkaline Lysis for Mini Prep
The DNA can then be used in a test digest using between 2 and 10 ul depending upon the origin of replication e.g. pUC19 use 2 ul and pACYC plasmids use 10 ul.
 * 1) Use a 3 - 5 ml culture either grown ON or day growth
 * 2) spin and resuspend in 300ul of Sol.I+RNase
 * 3) add 600 ul fresh Sol.II (2ml NaOH (1M) 1ml 10%SDS and 7 ml water)
 * 4) 5 min RT then add 250 ul Sol III - SHAKE RIGOROUSLY!
 * 5) spin 20 min
 * 6) 1ml supernatant into new tube and add 600 ul Isopropanol
 * 7) 15 min spin and aspirate
 * 8) add 500 ul 70% ethanol and spin for 5 min
 * 9) aspirate and speed vac resuspending in 50 ul water

Solution I (can be stored at RT until Rnase A added) you can make it up to 500 ml
 * 50 mM Glucose
 * 25 mM Tris.Hcl (pH 8.0)
 * 10 mM EDTA (pH 8.0)

Add 250 ul of RNase A to 50 ml of Sol. I then store at 4°C

Solution III (for 100 ml)
 * 5M Potassium acetate: 60ml
 * glacial acetic acid: 11.5 ml
 * H2O: 28.5 ml

Freezing Strains into the TPA Collection

 * In general
 * This protocol is designed to aid you in freezing strains
 * Solutions required:
 * 2.0ml screw cap tubes (sterile)
 * DMSO (sterile)


 * Before Freezing
 * Make sure you have all your strains freshly streaked out on plates
 * Assign each strain to be frozen a TPA number completing the strain book FIRST (P.T.O for examples and help)
 * Pick a single colony and inoculate 5ml LB or PYE +/- antibiotic (if necessary) culture
 * Incubate cultures overnight at the correct temperature


 * Freezing
 * Label the correct number of screw cap tubes to freeze strains in duplicate
 * Add 150ul sterile DMSO to each tube using fresh tips each time
 * Add 1.5ml of each culture to each tube, mixing by pipetting up and down
 * Place in correct boxes in -80 degree Celsius freezer

NOTE: Box numbers start at the top left hand corner and go vertically down


 * Once in Freezer go back to strain book and date and initial all strains frozen.