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From 2013.igem.org

Contents 1 Chemically competent E.coli cells: CaCl2 method 1.1 Procedure 1.2 Buffers and solutions 2 Chemically competent E.coli cells: Inoue method 2.1 Procedure 2.2 Buffers and solutions 3 Plasmid DNA isolation 3.1 Procedure 3.2 Buffer and solution 4 Colony PCR for Streptomyces 4.1 Pretreatment of Streptomyces 4.2 PCR mixture 4.3 Cycling instruction 5 PCR Protocol for Taq DNA Polymerase with Standard Taq Buffer 5.1 Reaction set up 5.2 Thermocyclingconditions for a routine PCR 6 PCR clean-up 7 DNA extraction from agarose gels

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!

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

Buffers and solutions

Risk assessment for pH electrode and preparation of buffers

• Growth medium
  • LB 25g/l
• Frozen Storage Buffer (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)

Chemically competent E.coli cells: Inoue method

Procedure

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

1. Pick a single colony from a freshly transformed plate (after overnight growth @ 37 °C)
2. Transfer the colony to 25 ml growth medium in a sterile 250 ml erlenmeyer
3. Incubate the culture @ 37°C for 6 – 8 hrs under vigourous shaking (250 – 300 rpm)
4. Prepare 3 1L flasks with 250 ml growth medium in each
5. Inoculate the flasks with 10, 4 or 2 ml of the dayculture -> you create 3 different starting optical densities.
6. Incubate the cultures @ 18-22°C overnight under moderate shaking (180 – 220 rpm)
7. Monitor the OD600nm until it reaches 0.55
8. Place cells in an ice-water bath to cool them down quickly (-> swirl occasionally, keep them in for approx 10min)
9. Centrifuge cells @4°C for 10min at 2500g
10. Pour off supernatant – make sure all remaining droplets are removed
11. Resuspend gently (swirl !) in 80 ml icecold inoue transformation buffer
12. Centrifuge cells @4°C for 10min at 2500g
13. Pour off supernatant – make sure all remaining droplets are removed
14. Resuspend gently (swirl !) in 20 ml icecold inoue transformation buffer
15. Add 1.5 ml 100% DMSO – mix by swirling
16. Store whole on ice for approx 10 minutes
17. Aliquot as quickly as possible 100 – 200 microliter aliquots into 1.5 ml tubes (precooled on ice) and snapfreeze them into a liquid N2 bath

Buffers and solutions

• Growth medium
• Inoue transformation buffer

Reagent	Final concentration (mM)	Amount per liter
MnCl2	55	10.88g (from MnCl2*4H2O)
CaCl2	15	2.20g (from CaCl2*2H2O)
KCl	250	18.65g (from KCl)
PIPES	10	20ml (from 0.5M stock solution)
H2O	to 1 liter

Filter sterilize with a 0.45 µmeter nalgene filter

Risk assessment for pH electrode and preparation of buffers

• 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 µmeter nalgene filter
  • Aliquot (5 times) and store at -20°C

Plasmid DNA isolation

Procedure

Risk assessment for plasmid DNA purification kit

1. Bring 1.5 ml culture in an eppendorf, centrifuge for 1min with maximum speed
2. Pour away the supernatant
3. Bring another 1.5 culture into the same eppendorf, centrifuge for 1min and pour away supernatant
4. Resuspend the pellet with 200µl GTE-solution we made earlier
5. Add 4µl RNase A (10mg/ml)
6. Add 400µl premade solution (contain 0.2M NaOH and 1%SDS in sterile water)
7. Mix them well, place on ice for 5min
8. Add 300µl ice cold 7.5 ammonium acetate, vortex for 10s, place on ice for 5mins
9. Centrifuge for 5min with 13000 rpm
10. Bring the supernatant into a new eppendorf
11. Centrifuge this supernatant for a second time (5min, 13000rpm) and bring the supernatant in a new eppendorf
12. Add isopropanol to the supernatant (60% in volume of the supernatant), left in room temp. for 5min
13. 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
14. Add 1ml of cold 70% ethanol to the pellet, invert 5 times
15. Centrifuge 3min with 13000rpm
16. Remove supernatant, the droplet on the tube wall can be removed by tissue
17. Let the pellet dry
18. Add 50µl elution buffer (or sterile water) to the pellet

Buffer and solution

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

• IPTG stock solution
  • 238 mg in 10 ml AD
  • Filter sterilize
  • Split into 1 ml aliquots
  • 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:

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

PCR mixture

Risk assessment for PCR

Components	Amount
2x fusion master mix (add in the end)	25µl
forward primer (final conc. 0.5µM)	1.25µl (of 20µM stock)
reverse primer (final conc. 0.5µM)	1.25µl (of 20µM stock)
template DNA	1µl
DMSO (recommended for high GC content)	1.5µl
H2O (PCR certified, no contamination)	add to final volume of 50µl

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

Cycling instruction

Step	Temperature	Time
1	95°C	6'
2 cycle 29x	95°C 55°C 72°C	30" 30" 45"
3	72°C	10'
4	12°C	infinite/hold

PCR Protocol for Taq DNA Polymerase with Standard Taq Buffer

Reaction set up

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

Components	25μl reaction	50μl reaction	Final concentration
10X Standard Taq Reaction Buffer	2.5µl	5µl	1X
10 mM dNTPs	0.5µl	1µl	200µM
10 µM Forward Primer	0.5µl	1µl	0.2 µM (0.05–1 µM)
10 µM Reverse Primer	0.5µl	1µl	0.2 µM (0.05–1 µM)
template DNA	variable	variable	<1,000 ng
Taq DNA Polymerase	0.125 µl	0.25 µl	1.25 units/50 µl PCR
Nuclease-free water	to 25 µl	to 50 µl

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.

Thermocyclingconditions for a routine PCR

Step	Temperature	Time
Initial denaturation	95°C	30"
30 cycles	95°C 48-65°C 68°C	15-30" 15-60" 1min/kb
Final extension	68°C	5'
Hold	12°C	infinite/hold

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.