Team:KU Leuven/Protocols

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Revision as of 11:20, 15 July 2013

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Chemically competent E.coli cells: CaCl2 method

Procedure

Perform every action on ice – also when resuspending your cells! Do not shock freeze (liqN2) – just transfer from ice to -80°C! Work sterile

• Inoculate 3ml growth medium with your cells of choice (DH5alpha or TOP10 for plasmid maintenance & cloning)
• Grow overnight at 37°C with sufficient aeration
• Inoculate 100 ml LB with 1 ml of overnight culture
• Grow at 37°C to an Optical Density at 600nm of approx 0.5 to 0.8 (usually 2-3 hrs)
• Centrifuge cells (3700-4000 rpm 4°C 12min – sterile 50ml tube)
• Re-suspend pellet on ice with FSB to 15 ml (cold) for each 100 ml pellet
• Incubate cells 10min on ice
• Centrifuge cells (3700 – 4000 rpm 4°C 10min)
• Re-suspend pellet on ice in 4-8 ml FSB (cold) for each 100 ml pellet
• Aliquot cells appropriately (200-400 µl aliquots) and freeze aliquots at -80°C

Buffers and solutions

1. Growth medium
   • LB 25g/l
2. FSB
   • 10 mM Potassium Acetate
   • 10% glycerol
   • 10 mMKCl
   • 50 mM CaCl2
   • Check pH – must be around 6.2 – if need be adjust with AcAc (HCl) or KOH
   • Buffer should be filter-sterilized (0.45 micrometer filter) but has been autoclaved also

Chemically competent E.coli cells: Inoue method

Procedure

Perform every action on ice – also resuspending your cells !! Work sterile

• Pick a single colony from a freshly transformed plate (after overnight growth @ 37 degrees)
• Transfer the colony to 25 ml growth medium in a 250 ml erlenmeyer (sterile !)
• Incubate the culture @ 37°C for 6 – 8 hrs under vigourous shaking (250 – 300 rpm)
• Prepare 3 1L flasks with 250 ml growth medium in each
• Inoculate the flasks with 10, 4 or 2 ml of the dayculture -> you create 3 different starting optical densities.
• Incubate the cultures @ 18-22°C overnight under moderate shaking (180 – 220 rpm)
• Monitor the OD600nm until it reaches 0.55
• Place cells in an ice-water bath to cool them down quickly (-> swirl occasionally, keep them in for approx 10min)
• Harvest cells @4°C for 10min at 2500g
• Pour off supernatant (to biological waste) – make sure all remaining droplets are removed
• Resuspend gently (swirl !) in 80 ml icecoldinoue transformation buffer
• Harvest cells @4°C for 10min at 2500g
• Pour off supernatant (to biological waste) – make sure all remaining droplets are removed
• Resuspend gently (swirl !) in 20 ml icecoldinoue transformation buffer
• Add 1.5 ml 100% DMSO – mix by swirling
• Store whole on ice for approx 10 minutes
• Aliquot as quickly as possible 100 – 200 microliter aliquots into 1.5 ml tubes (precooled on ice) and snapfreeze them into a liqN2 bath

Buffers and solutions

• Growth medium
• Inoue transformation buffer

Filter sterilize with a 0.45 micrometernalgene filter

• Stock 0.5M PIPES (piperazine-1,2-bis[2-ethanesulfonic acid]) pH 6.7
  • Dissolve 15.1g PIPES in 80ml MilliQ H2O
  • Adjust pH to 6.7 with 5M KOH
  • Bring volume to 100 ml with MilliQ H2O
  • Filter sterilize with a 0.45 micrometernalgene filter
  • Aliquot (5 times) and store at -20°C

Plasmid DNA isolation

Procedure

• bring 1.5 ml culture in an eppendorf, centrifuge for 1min with maximum speed
• pour away the supernatant
• bring another 1.5 culture into the same eppendorf, centrifuge for 1min and pour away supernatant
• re-suspend the pellet with 200µl GTE-solution we made earlier
• add 4µl RNase A (10mg/ml)
• add 400µl premade solution (contain 0.2M NaOH and 1%SDS in sterile water)
• mix them well, place on ice for 5min
• add 300µl ice cold 7.5 ammonium acetate, vortex for 10s, place on ice for 5mins
• centrifuge for 5min with 13000 rpm
• bring the supernatant into a new eppendorf
• centrifuge this supernatant for a second time (5min, 13000rpm) and bring the supernatant in a new eppendorf
• add isopropanol to the supernatant (60% in volume of the supernatant), left in room temp. for 5min
• centrifuge for 10min with 13000rpm, immediately remove the supernatant, keep the transparent pellet in the tube, put the tube upside down on a tissue to dry it
• add 1ml of cold 70% ethanol to the pellet, invert 5 times
• centrifuge 3min with 13000rpm
• remove supernatant, the droplet on the tube wall can be removed by tissue
• let the pellet dry
• add 50µl elution buffer (or sterile water) to the pellet

Buffer and solution

1. GTE-solution
   • 50 mM glucose
   • 10 mM EDTA
   • 25 mM Tris-Cl (pH 8.0)
   • 4 mg/ml lysozyme

IPTG stock solution

• 238 mg in 10 ml AD
• Filter sterilize
• Split into 1 ml aliquots
• Store in -20 freezer

OR

• 23.8 mg in 1 ml AD
• Filter sterilize
• Store in -20 freezer

Final concentration/work concentration in agar plates = 0.1mM – 1 mM Sigma recommends 0.2mM for blue-white screening Thermo Scientific recommends 0.1mM

Colony PCR for Streptomyces

Pretreatment of Streptomyces

Because of the fact that Streptomyces is gram negative bacteria, we performed 4 ways to extract its genome:

• 8microwave Streptomyces for 4mins
• mix Streptomyces with water and 0.2%SDS, 4min microwave
• mix streptomyces with 1% SDS, 4min microwave
• mix streptomyces with TE buffer, 0.2% SDS, 4min microwave

PCR mixture

Keep tubes on ice at all times! Be sure to put Phusion Master Mix immediately back at -20!

Cycling instruction

PCR Protocol for Taq DNA Polymerase with Standard Taq Buffer

Reaction set up

We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (95°C).

Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid. Transfer PCR tubes from ice to a PCR machine with the block preheated to 95°C and begin thermocycling.

Thermocyclingconditionsfor a routine PCR

PCR clean-up

(source: NucleoSpin® Gel and PCR Clean-up) This is used for PCR clean-up as well as DNA concentration and removal of salts, enzymes, etc. from enzymatic reactions (SDS<0.1%)

1. Adjust DNA binding condition: mix 1 volume of sample with 2 volumes of buffer NTI (eg. mix 100µl PCR reaction and 200µl buffer NTI).
2. Binding DNA: place a PCR clean-up column into a collection tube (2ml) and load up to 700µl sample, centrifuge for 30s at 11000g, discard flow-through and place the column back into the collection tube.
3. Wash silica membrane: add 600µl buffer NT3 to the column, centrifuge for 30s at 11000g, discard flow-through and place the column back into the collection tube. Repeat the washing again.
4. Dry silica membrane: centrifuge for 1min at 11000g to remove buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.
5. Elute DNA: place the column into a new 1.5ml microcentrifuge tube, add 50µl buffer NE and incubate at room temperature for 1min, centrifuge for 1min at 11000g.

DNA extraction from agarose gels

(source: NucleoSpin® Gel and PCR Clean-up)

1. Excise DNA fragment/solubilize gel slice: take a clean scalpel to excise the DNA fragment from an agarose gel, remove all excess agarose. For each 100mg of agarose gel < 2% add 200µl buffer NTI, for gels containing > 2% agarose, double the volume of buffer NTI. Incubate sample for 5-10min at 50°C, vortex the sample briefly every 2-3min until the gel slice is completely dissolved.
2. Binding DNA: place a PCR clean-up column into a collection tube (2ml) and load up to 700µl sample, centrifuge for 30s at 11000g, discard flow-through and place the column back into the collection tube.
3. Wash silica membrane: add 700µl buffer NT3 to the column, centrifuge for 30s at 11000g, discard flow-through and place the column back into the collection tube. Repeat the washing again.
4. Dry silica membrane: centrifuge for 1min at 11000g to remove buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.
5. Elute DNA: place the column into a new 1.5ml microcentrifuge tube, add 15-30µl buffer NE and incubate at room temperature for 1min, centrifuge for 1min at 11000g.