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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 Colony PCR for Streptomyces 3.1 Pretreatment of Streptomyces 3.2 PCR mixture 3.3 Cycling instruction 4 Digestion and ligation 4.1 Consumables and equipment 4.2 Digestion 4.3 Ligation 5 DNA extraction from agarose gels 6 Grow electrocompetent cells 6.1 Materials 6.2 Special Equipment 6.3 Method 7 Isolation of plasmid DNA from E. coli (mini prep) 8 Nanodrop protocol 9 PCR clean-up 10 PCR Protocol for Taq DNA Polymerase with Standard Taq Buffer 10.1 Reaction set up 10.2 Thermocyclingconditions for a routine PCR 11 Plasmid DNA isolation 11.1 Procedure 11.2 Buffer and solution 12 qRT-PCR Protocol 12.1 Sample preparation 12.2 mRNA isolation 12.3 Create cDNA 12.4 qPCR

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 OD 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 mM KCl
  • 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)

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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 at 37 °C)
2. Transfer the colony to 25 ml growth medium in a sterile 250 ml erlenmeyer
3. Incubate the culture at 37°C for 6 – 8 hrs under vigorous 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 at 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 10 min)
9. Centrifuge cells at 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 at 4°C for 10 min 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

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Colony PCR for Streptomyces

Pretreatment of Streptomyces

Because of the fact that Streptomyces are Gram-positive bacteria with a thick peptidoglycan layer, we performed 4 ways to pretreat the cells for colony PCR (all pretreatments gave positive results in the end):

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

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

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Digestion and ligation

Consumables and equipment

• Restriction enzymes (EcoRI, Xbal, Spel, Pstl), NEBuffer 2.1
• 10x T4 DNA ligase Reaction Buffer, T4 DNA Ligase
• Keep all enzymes on ice; make sure buffers have no precipitation
• H20
• Small PCR Tubes or eppendorfs
• 2 µl, 200 µl pipette tips
• Destination plasmid as purified DNA
• Upstream and downstream part as purified DNA
• 2 µl and 20 µl pipette
• Heat block 37° and 80°C
• Timer
• Rack for small PCR tubes
• -20°C freezer + freeze box

Digestion

• Mark PCR tubes or eppendorfs

   a. U= upstream part : E + S restriction enzymes
   b. D= downstream part : X + P restriction enzymes
   c. P= plasmid (destination) : E + P restriction enzymes
   d. NB: if only one part for insertion insert I= Insert : E + P restriction enzymes

• In each tube 500 ng DNA for digestion + H20 until total volume is 43 µl
• Add 5 µl of NEBuffer 2.1 to each tube
• Add 1 µl of first restriction enzyme
• Add 1 µl of the second restriction enzyme TOTAL VOLUME = 50 µl
• Mix well by flicking each tube
• Incubate at 37°C for 20 min. (officially 15 min)
• Incubate at 80°C for 20 min.

   a. OPTIONAL: run 10-20 µl on 1% agarose gel and look for expected bands as confirmation
   b. OPTIONAL: store at -20°C or proceed to ligation immediately

Ligation

• Add 13 µl of H2O to a 200 µl PCR tube or eppendorf
• Add 2 µl of each part you want to ligate
• Add 2 µl of 10X T4 DNA Ligase Reaction Buffer to the tube
• Add 1 µl of the T4 DNA Ligase to the tube TOTAL VOLUME = 20 µl
• Mix well by flicking each tube
• Incubate at room temperature for 10 min

   a. Incubate at 80°C for 20 min. 
   b. Store the ligation mix at -20°C or proceed immediately to the transformation step.

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DNA extraction from agarose gels

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

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 5-10 min at 50°C, vortex the sample briefly every 2-3 min 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 1 min, centrifuge for 1 min at 11000g.

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Grow electrocompetent cells

(source: adapted from openwetware.org)

Materials

• GYT (glycerol, yeast extract, tryptone)
  • 10%(v/v) glycerol
  • 0.125% (w/v) yeast extract
  • 0.25% (w/v) tryptone

• DI water
• 10% Glycerol

Special Equipment

• Centrifuge
• Ice water bath
• Liquid nitrogen

Method

Important: All steps in this protocol should be carried out aseptically

• Inoculate: Prepare flask containing 5 ml of LB medium. Pick up a single colony of cells from plate (using a sterile toothpick) and swirl around inside flask. Incubate the culture overnight at 37°C with vigorous aeration (250 pm in a rotary shaker).

• Dilute and incubate: Inoculate two aliquots of 495 ml of prewarmed LB medium in separate 2-liter flasks with 5 ml of the overnight bacterial culture. Incubate the flasks at 37°C with agitation (300 cycles/min in a rotary shaker). Measure the OD-600 every twenty minutes (this step will take around 1.5-2 hrs). (or judge the OD by eyes to avoid always taking the sample to disturb the growth as well as avoiding the contamination)

• Rapidly cool culture: Once the OD-600 of the culture reaches 0.6-1.0 (Molecular Cloning recommends 0.4), rapidly transfer the flasks to the pre-made ice-water bath for 15-30 minutes. Swirl the culture occasionally to ensure that cooling occurs evenly. In preparation for the next step, place the centrifuge bottles in the ice-water bath as well.

Note: After this point, do not let your cells warm up past 4°C always keep on ice

Note: When harvesting cells by decanting, be very careful to not disturb the pellet-- this could result in a much lower yield. If necessary, aspirate instead of decant the supernatant. Get someone to show you how to aspirate. Also, if the pellet seems loose, sometimes it is helpful to re-spin the cells down.

• Centrifuge 1: Transfer the cultures to ice-cold centrifuge bottles. Harvest the cells by centrifugation at 1000g (2500 rpm) for 15 minutes at 4°C. Decant the supernantant and resuspend the cell pellet in 20 ml of ice-cold 10% glycerol. Note: I think this should be done for each of the two 500ml cultures, i.e this is a 1:1 resuspension rather than a concentration by a factor of 2 BC.

• Centrifuge 2 (10% glycerol): Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in 20 ml ice-cold 10% glycerol.

• Centrifuge 3 (10% glycerol): Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in 10 ml ice-cold 10% glycerol.

• Centrifuge 4 (10% glycerol): Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. Carefully decant the supernatant and use a Pastteur pipette attached to a vacuum line to remove any remaining drops of buffer.

• Resuspend in GYT: Resuspend in 1 ml ice cold GYT. This is best done by gently swirling rather pipetting or vortexing.

• Test for arcing: Transfer 40 ul of the suspension to an ice-cold electroporation cuvette (0.1-0.2 cm gap, on middle shelf next to electroporator) and test whether arcing occurs when an electrical discharge is applied. Place the cuvette in the green holder attached to the machine. Go to option 4, Pre-set protocols; choose bacterial; choose the correct choice for your size cuvette, probably the first option for a .1 cm cuvette. If arcing occurs, wash the remainder of the cell suspension once more with ice-cold GYT medium to ensure that the conductivity of the bacterial suspension is sufficiently low (<5 mEq). (or checking the pulse time, if the pulse time < 4, redo the wash, if the pulse time > 4, it's ok)

• Storage: Store cells at -80°C until they are required for use. For storage, dispense 40 ul aliquots of the cell suspension into sterile, ice-cold .5 ml microcentrifuge tubes, drop into a bath of liquid nitrogen and transfer to a -80°C freezer. To remove the tubes from the liquid nitrogen bath, bring out into the hall along with a storage box, and pour the tubes and liquid nitrogen into the box. Once all the tubes are out, close the box most of the way and let the liquid run out into the hallway. Try not to do this in the very center of the walkway!

• To use frozen cells: Remove an appropriate number of aliquots of cells from the -80°C freezer. Thaw the tubes on ice.

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Isolation of plasmid DNA from E. coli (mini prep)

(source: NucleoSpin® plasmid - Macherey-Nagel)

Nanodrop protocol

Nanodrop can be used to measure the DNA, RNA and protein Measure the concentration and purity of extracted DNA using absorbance (using the automated nanodrop machine!)

Method:

1. Log onto computer and select Nanodrop program from the desktop (ND 1000)
2. To clean Nanodrop machine wipe pedestal and top and add 3 µl of water to nib of pedestal. Press blank.
3. Wipe the water off, to initialise/equalizen the equipment add 3 μl of the elution buffer [EB] used in the sample and press blank. Set to DNA-50 for DNA.
4. Wipe to remove buffer and apply 3 μl of sample to nib. Press measure.
5. If dealing with multiple samples, clean the equipment with water at regular intervals (about every 10 samples).
6. After measurements, clean the equipment with 3 μl of water on the spectrometer and press blank. Wipe and log off.

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PCR clean-up

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

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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

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Plasmid DNA isolation

Procedure

Risk assessment for plasmid DNA purification kit

1. Bring 1.5 ml culture in an eppendorf, centrifuge for 1 min with maximum speed
2. Pour away the supernatant
3. Bring another 1.5 ml culture into the same eppendorf, centrifuge for 1 min 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 (contains 0.2M NaOH and 1%SDS in sterile water)
7. Mix them well, place on ice for 5 min
8. Add 300µl ice cold 7.5 M ammonium acetate, vortex for 10s, place on ice for 5 mins
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), leave @ room temp. for 5 min
13. Centrifuge for 10 min with 13000 rpm, immediately remove the supernatant, keep the transparent pellet in the tube, put the tube upside down on a tissue to dry it
14. Add 1 ml of cold 70% ethanol to the pellet, invert 5 times
15. Centrifuge 3 min with 13000 rpm
16. Remove supernatant, the droplet on the tube wall can be removed by tissue
17. Let the pellet dry
18. Add 50 µl 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

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qRT-PCR Protocol

General experiment: 3 biological repeats of stationary phase cultures 16hrs growth.

Sample preparation

1. Inoculate fresh LB plate with the strain harbouring the MS brick
2. Isolate plasmid DNA, MS brick
3. Nanodrop plasmid (minimum concentration 20 ng/µl) = 155 ng/µl
4. Design primers for pcha/pchb and for bsmt1 using Primer express. Primer sequence can be found in document “Genes_qPCR_primers_IC.docx”.
5. Inoculate E. Coli with Methylsalicylate brick (MIT 2006) on LB plate.
6. Add 5 µl of Kanamicin (50 mg/ml) and 10 µl of IPTG (100 mM) to 5 ml of LB medium (tubes).
7. Incubate 16h at 37 °C under shaking conditions.
8. Measure OD of 3 cultures.
9. Account for differences in OD: ex. If you have an OD of 1 take 1 ml of this sample, if you have an OD of 0,8 take 1,2 ml.
10. Add 1/5 volume of stopsolution (95% ethanol, 5% phenol).
11. Freeze 5 minutes in liquid Nitrogen.
12. Centrifuge needed amount of cells.

mRNA isolation

For mRNA isolation use the Promega SV total RNA isolation kit ([http://be.promega.com/resources/protocols/technical-manuals/0/sv-total-rna-isolation-system-protocol/?origUrl=http%3a%2f%2fwww.promega.com%2fresources%2fprotocols%2ftechnical-manuals%2f0%2fsv-total-rna-isolation-system-protocol%2f Promega SV total RNA isolation protocol])

1. Section 8.C (Isolation of RNA from Gram-positive and gram-Negative bacteria)
2. Section 4.E (RNA Purification by Centrifugation) We did not do step 4 and 5 (addition of DNase) but instead used the DNase protocol of [http://products.invitrogen.com/ivgn/product/AM2238 Ambion Turbo DNase]:

For 100 µl of RNA:

1. 1. Add 10 µl 10x buffer. Add 1µl Turbo DNase and mix gently.
   2. Put on 37 °C for 20 to 30 min.
   3. Add 1µl of Turbo DNase.
   4. Put on 37 °C for 20 to 30 min.
   5. Add 20 µl of DNase inactivation reagent and mix well.
   6. Put on room temperature for 5 min. Mix occasionally by flicking the tube
   7. Centrufige on 10000 x g for 2 min.
   8. Transfer the RNA to a new tube.

Create cDNA

Use the Fermentas Revert aid H kit ([http://www.thermoscientificbio.com/reverse-transcription-rtpcr-rtqpcr/revertaid-h-minus-first-strand-cdna-synthesis-kit/ Fermentas Revert aid H protocol]) The protocol for RT-PCR (I. First Strand Synthesis) was followed

qPCR

qPCR kit was not yet decided.