Team:Kyoto/Protocols

From 2010.igem.org

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(Measurement the time that cell lysis becomes obvious)
 
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{{:Team:Kyoto/Header}}
{{:Team:Kyoto/Header}}
-
==Transformation==
+
==Protocols==
 +
===Index===
 +
* [[#Construction|Construction]]
 +
** [[#Solubilization of Antibiotics|Solubilization of Antibiotics]]
 +
** [[#Media|Media]]
 +
** [[#Transformation|Transformation]]
 +
** [[#Miniprep|Miniprep]]
 +
** [[#Restriction Digestion|Restriction Digestion]]
 +
** [[#PCR|PCR]]
 +
** [[#Electrophoresis|Electrophoresis]]
 +
** [[#Gel Extraction|Gel Extraction]]
 +
** [[#Ligation|Ligation]]
 +
** [[#Dephosphorylation|Dephosphorylation]]
 +
** [[#Sequence|Sequence]]
 +
** [[#Ethanol Precipitation|Ethanol Precipitation]]
 +
* [[#Measurement|Measurement]]
 +
** [[#Promoter Activity (from 24 August to 15 September)|Promoter Activity (from 24 August to 15 September)]]
 +
** [[#Promoter Activity (from 16 September)|Promoter Activity (from 16 September)]]
 +
** [[#GFP Fluorescence (from 24 August to 17 October)|GFP Fluorescence (from 24 August to 17 October)]]
 +
** [[#Promoter Activity and GFP Fluorescence (from 18 October)|Promoter Activity and GFP Fluorescence (from 18 October)]]
 +
** [[#Measurement of the actitvity of inducible lytic system|Measurement of the actitvity of inducible lytic system]]
-
==Miniprep==
+
===Construction===
-
* Use QIAprep Spin Miniprep Kit Cat. No. 27104 by QIAGEN
+
====Solubilization of Antibiotics====
-
# Pick a single colony from a freshly streaked selective plate and inoculate a culture of about 3ml LB medium containing the appropriate selective antibiotic.
+
# Mix the following (Final concentration is 50mg/mL).
 +
#* Ampicillin:
 +
#*; Ampicillin: 1.0g
 +
#*; MilliQ: 20mL
 +
#* Kanamycin:
 +
#*; Kanamycin: 0.5g
 +
#*; MilliQ: 10mL
 +
# Dispense 1.1mL of the solution into 1.5mL tubes.
 +
# Store in the -20℃ freezer.
 +
 
 +
====Media====
 +
=====LB Media=====
 +
# Wash a graduated cylinder with MilliQ.
 +
# Add the following to about 180mL of MilliQ in the graduated cylinder:
 +
#; Bacto-yeast extract: 1.0g (0.5w/v%)
 +
#; Tryptone: 2.0g (1.0w/v%)
 +
#; NaCl: 2.0g (1.0w/v%)
 +
#; 1N NaOH: 200µL
 +
# Seal the graduated cylinder by a Parafilm.
 +
# Dissolve the mixture by inverting the graduated cylinder.
 +
# Adjust volume to 200mL by adding more MilliQ.
 +
# Add the media solution to a 200mL Erlenmeyer flask.
 +
# (To prepare solid media, add 2.4g (1.2w/v%) of agar to the flask.)
 +
# Wrap the tops of the flasks with aluminum foil.
 +
# Place a small piece of auto clave tape on one of the flasks.
 +
# Autoclave the media on liquids.
 +
# Store at room temperature.
 +
# To pour plates, melt the solid media in the microwave, then place flask in water bath to bring media to 50℃.
 +
# Add 200µL of antibiotic
 +
#; Ampicillin: 100µg/mL
 +
#; Kanamycin: 50µg/mL
 +
# (Add 0.5 - 1.0w/v% Glucose for protein expression.)
 +
 
 +
=====Supplemented M9 Media=====
 +
# Wash a graduated cylinder with MilliQ.
 +
# Add the following to about 180mL of MilliQ in the graduated cylinder:
 +
#; Na2HPO4: 1.2g (0.6w/v%)
 +
#; KH2PO4: 0.6g (0.3w/v%)
 +
#; NaCl: 0.1g (0.05w/v%)
 +
#; NH4Cl: 0.2g (0.1w/v%)
 +
# Seal the graduated cylinder by a Parafilm.
 +
# Dissolve the mixture by inverting the graduated cylinder.
 +
# Adjust volume to 200mL by adding more MilliQ.
 +
# Add the medium solution to a 200mL Erlenmeyer flask.
 +
# Wrap the tops of the flasks with aluminum foil.
 +
# Place a small piece of auto clave tape on one of the flasks.
 +
# Autoclave the media on a liquids.
 +
# Cool at room temperature.
 +
# When it become as cool as you can hold it,Add the following to it:
 +
#; MgSO4: 1.0mM(final cocentration)
 +
#; CaCl2: 0.1mM(final concentration)
 +
#; thiamine hydrochloride: 0.001%(final concentration)
 +
#; casamino acids: 0.2w/v%
 +
# Add 200µL of antibiotic:
 +
#; Ampicillin: 100µg/mL
 +
#; Kanamycin: 50µg/mL
 +
# Add 0.4w/v% Glucose.
 +
 
 +
====Transformation====
 +
# Unfreeze conpitent cells on ice.
 +
# Dry a plate by letting the plate upside down and partly open in incubator.
 +
# Add 1µL DNA solution and 20µL compitent cells to 1.5mL tube, let stand for 30min on ice. If few colony is observed, increase the amount of the compitent cells or DNA, but make the amount of DNA not to get over that of the compitent cells.
 +
# Heatshock for 60s at 42℃.
 +
# Let stand for 2min on ice.
 +
# Culture for 1h in preculture medium (LB or SOC medium), and plate by using spreader. Do not heat spreader too much because e.coli will dead for heat.
 +
 
 +
====Miniprep====
 +
# Use QIAprep Spin Miniprep Kit Cat. No. 27104 by QIAGEN
 +
# Pick a single colony from a freshly streaked selective plate and inoculate a culture of about 3mL LB medium containing the appropriate selective antibiotic.
# Incubate at 170rpm for 8h at 37℃ with vigorous shaking.
# Incubate at 170rpm for 8h at 37℃ with vigorous shaking.
# Transfer a half of the culture to a tube.
# Transfer a half of the culture to a tube.
-
# Harvest the bacterial cells by centrifugation at 14,000 x g for 1min at 4℃. Remove the medium by decanting.
+
# Harvest the bacterial cells by centrifugation at 14,000g for 1min at 4℃. Remove the medium by decanting.
# Transfer the half of the culture to same tube and harvest as same. Remove the medium by pippetting.
# Transfer the half of the culture to same tube and harvest as same. Remove the medium by pippetting.
-
# Resuspend pelleted bacterial cells in 250µl Buffer P1 and mix thoroughly by pippeting.
+
# Resuspend pelleted bacterial cells in 250µL Buffer P1 and mix thoroughly by pippeting.
-
# Add 250µl Buffer P2 and mix thoroughly by inverting the tube gently 4-6 times.
+
# Add 250µL Buffer P2 and mix thoroughly by inverting the tube gently 4-6 times.
-
# Add 350µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
+
# Add 350µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
-
# Centrifuge for 10min at 14,000 x g at 4℃.
+
# Centrifuge for 10min at 14,000g at 4℃.
# Apply the supernatants from step 10 to the QIAprep spin column by pipetting.
# Apply the supernatants from step 10 to the QIAprep spin column by pipetting.
# Centrifuge for 10s in a table-top microcentrifuge. Discard the flow-through.
# Centrifuge for 10s in a table-top microcentrifuge. Discard the flow-through.
-
# Wash the QIAprep spin column by adding 0.5ml Buffer PB and centrifuging for 10s in a table-top microcentrifuge. Discard the flow-through.
+
# Wash the QIAprep spin column by adding 0.5mL Buffer PB and centrifuging for 10s in a table-top microcentrifuge. Discard the flow-through.
-
# Wash QIAprep spin column by adding 0.65ml Buffer PeE and centrifuging fo 10s in a table-top microcentrifuge.
+
# Wash QIAprep spin column by adding 0.65mL Buffer PeE and centrifuging fo 10s in a table-top microcentrifuge.
# Discard the flow-through, and centrifuge for and additional 1min to remove residual wash buffer.
# Discard the flow-through, and centrifuge for and additional 1min to remove residual wash buffer.
-
# Place the QIAprep column in a clean tube. To elute DNA, add 50µl water to the center of each QIAprep spin column, let stand for 1min, and centrifuge for 1min.
+
# Place the QIAprep column in a clean tube. To elute DNA, add 50µL water to the center of each QIAprep spin column, let stand for 1min, and centrifuge for 1min.
# Discard the QIAprep spin column.
# Discard the QIAprep spin column.
# Measure the concentration of DNA by using eppendorf BioPhotometer plus.
# Measure the concentration of DNA by using eppendorf BioPhotometer plus.
Line 24: Line 112:
# Agarose Gel Electrophoresis for Confirmation.
# Agarose Gel Electrophoresis for Confirmation.
-
==PCR==
+
====Restriction Digestion====
-
# Dilute template DNA. If the concentration of DNA is 2-100 ng/µL, transfer 1µl to a clean tube and add 99µl Milli-Q.
+
# Use EcoRI, XbaI, SpeI, PstI, DpnI(NEB)
-
# Dilute Primer. If the concentration of Primer is XµM, dilute primer X timers and transfer 1µl to a clean tube and add 99µl Milli-Q.
+
# Mix the following.
-
# Make a solution as the below.
+
#; Sample: 5µL
 +
#; 10xBuffer: 1µL
 +
#; Restriction Enzyme: 0.1µL
 +
#; MilliQ: 3.9µL
 +
# Let stand for 2h at 37℃
 +
 
 +
====PCR====
 +
=====Standard PCR=====
 +
# Dilute template DNA. If the concentration of DNA is 2-100ng/µL, transfer 1µL to a clean tube and add 99µL MilliQ.
 +
# Dilute Primer. If the concentration of Primer is XµM, dilute primer X timers and transfer 1µL to a clean tube and add 99µL MilliQ.
 +
# Mix the following.
 +
## For use of KOD plus ver2:
 +
##; 25mM MgSO4: 3µL
 +
##; 2mM dNTPs: 5µL
 +
##; 10xBuffer for KOD plus ver.2: 5µL
 +
##; Template DNA  (5ng/µL): 5µL
 +
##; Primer Forward (10µM): 1.5µL
 +
##; Primer Reverse (10µM): 1.5µL
 +
##; KOD plus ver.2: 1µL
 +
##; MilliQ: 28µL
 +
##; Total: 50µL
 +
## For use of KOD FX:
 +
##; 2mM dNTPs: 10µL
 +
##; 2xBuffer for KOD FX: 25µL
 +
##; Template DNA: 5µL
 +
##; Primer Forward (10µM): 1.5µL
 +
##; Primer Reverse (10µM): 1.5µL
 +
##; KOD FX: 1µL
 +
##; MilliQ: 6µL
 +
##; Total: 50µL
 +
# (If amplification is not succeeded, try at 4.5 or 6.0µL 25mM MgSO4.)
# Let stand for 2min at 94℃.
# Let stand for 2min at 94℃.
# 25-40 cycles for 10s at 98℃, for 30s at Tm-5℃, and for 1min (1min for 1kb) at 68℃ (Tm is temparature at which primer will dissolve).
# 25-40 cycles for 10s at 98℃, for 30s at Tm-5℃, and for 1min (1min for 1kb) at 68℃ (Tm is temparature at which primer will dissolve).
Line 33: Line 151:
# Agarose Gel Electrophoresis for confirmation.
# Agarose Gel Electrophoresis for confirmation.
-
{|
+
=====Screening PCR=====
-
|+ Solution Component
+
# Mix the following (Do on PCR Bench).
-
|-
+
#; 10x PCR buffer (TAKARA): 40µL
-
!Component||Volume||Final Concentration
+
#; 2.5mM dNTP: 8µL
-
|-
+
#; Primer-1 (10pmol/µL): 8µL
-
|Water||28µl||
+
#; Primer-2 (10pmol/µL): 8µL
-
|-
+
#; Ex Taq HS (TAKARA): 1.6µL
-
|25mM MgSO4||3µl||1.5mM
+
#; MilliQ : 334µL (to total 400µL)
-
|-
+
# Dispense 25µL to 15 tubes.
-
|2mM dNTPs||5µl||0.2mM
+
# Pick a single colony and transfer it to each tubes.
-
|-
+
# Suspend the colony.
-
|10xBuffer for KOD plus ver.2||5µl||1x
+
# Let stand for 10min at 90℃.
-
|-
+
# 35 cycles for 30s at 94℃, for 30s 55℃, and for 1min at 72℃.
-
|Template DNA||5&micro;l||1ng << 50ng
+
# Let stand for 4min at 72&#x2103;.
-
|-
+
# Add 5mL Loading Buffer to the tubes.
-
|Primer Forward(10&micro;M)||1.5&micro;l||0.3&micro;M
+
# Agalose Gel Electrophoresis for confirmation.
-
|-
+
# Negative Control: Use nothing.
-
|Primer Reverse(10&micro;M)||1.5&micro;l||0.3&micro;M
+
# Positive Control: Use a colony that will yield a product with this primers.
-
|-
+
-
|KOD plus ver.2||1&micro;l||0.02U/&micro;l
+
-
|-
+
-
|Total||50&micro;||
+
-
|}
+
-
* * If amplification is not succeeded, try at 4.5 or 6.0&micro;l 25mM MgSO4.
+
-
==Gel Electrophoresis==
+
=====Mutagenesis (Point mutation, Deletion, Insertion)=====
 +
# Mix the following.
 +
#; 10xBuffer: 5&micro;L
 +
#; 2mM dNTP: 5&micro;L
 +
#; Primer Forward (10&micro;M): 1.5&micro;L
 +
#; Primer Reverse (10&micro;M): 1.5&micro;L
 +
#; Template Plasmid (50ng/&micro;L): 1&micro;L
 +
#; KOD plus ver.2: 1&micro;L
 +
#; MilliQ: 35&micro;L
 +
#; Total: 50&micro;L
 +
# Prepare control: instead of KOD plus ver.2, add 1&micro;L MilliQ.
 +
# Let stand for 2min at 94&#x2103;.
 +
# X cycles (1 cycle for 1kb) for 10s at 98&#x2103; and for Ymin (1min for 1kb) at 68&#x2103;.
 +
# Hold at 4&#x2103;.
 +
# Take 25&micro;L of the solutions into fresh tubes.
 +
# Add 1&micro;L ''Dpn''I (10U/&micro;L).
 +
# Let stand for 1h at 37&#x2103;.
 +
# Agarose gel electrophoresis, using 5&micro;L of the solution for confirmation.
 +
# Mix the following.
 +
#; Sample: 2&micro;L
 +
#; Ligation high: 5&micro;L
 +
#; T4 Polynucleotide Kinase (5U/&micro;L): 1&micro;L
 +
#; MilliQ: 7&micro;L
 +
#; Total: 15&micro;L
 +
# Let stand for 1h at 16&#x2103;.
 +
# Transformation, using 10&micro;L of the solution.
-
==Gel Extraction==
+
=====PCR Purification=====
-
* Use QIAquick Gel Extraction Kit
+
# Use QIAquick PCR Purification Kit Cat. No. 28104 by QIAGEN
 +
# Add BufferPB about 5 times as much as the product of PCR.
 +
# Apply the solution to the column.
 +
# Centrifuge for 1min at 1300rpm. Discard the flow-through. If the amount of the sample is much, repeat this step by same column.
 +
# Add 750&micro;L BufferPE and let stand for 2-3min. If the solution overflows, we decrease the amount of BufferPE.
 +
# Centrifuge for 1min and discard the through.
 +
# Centrifuge for additional 1min to remove residual buffer.
 +
# Place the column in a clean tube.
 +
# Add 10&micro;L BufferEB or MilliQ to the center of each column, let stand for 1min, and repeat this step.
 +
# Centrifuge for 1min at 13000rpm.
 +
# Discard the column.
 +
 
 +
====Electrophoresis====
 +
# Prepare 200mL of a 1.0% agarose solution:
 +
# Measure 2.0g agarose into a beaker.
 +
# Add 200mL 1xTAE buffer.
 +
# Wrap the top of the beaker with plastic wrap.
 +
# Punch a hole through the wrap with a pipette tip (To let out steam).
 +
# Dissolve the agarose by heating in microwave and swirling without boiling.
 +
# Allow the agarose to cool.
 +
# Pour the agarose solution into a gel tray on a gel maker.
 +
# If there is air bubbles, pushing them with a pipette tip.
 +
# Place comb in the maker.
 +
# Cover the maker with a plastic wrap.
 +
# Let stand for about 45min.
 +
# Remove the comb carefully.
 +
# Store in the Tupperware in the refrigerator.
 +
# Place the tray in electrophoresis chamber.
 +
# Cover the tray with 1xTAE buffer.
 +
# To prepare samples for electrophoresi, add 1&micro;L of 6x Loading Buffer for every 5&micro;L of DNA solution and mix well.
 +
# Load 6&micro;L of the DNA solution per well.
 +
# Electrophorese at 100V for about 30min until Loading Buffer have migrated approximately three-quaters of the gel.
 +
# Stain the gel in 0.5&micro;g/mL ethidium bromide for 20-30min.
 +
# Rinse the gel with MilliQ.
 +
# Place a plastic wrap on the transilluminator in the cabinet of Printgraph.
 +
# Place the gel on the transilluminator.
 +
# Turn on the transilluminator and confirm the position of the gel.
 +
# Shoot the picture.
 +
# Turn off the transilluminator.
 +
# Dispose of the gel.
 +
 
 +
====Gel Extraction====
 +
# Use QIAquick Gel Extraction Kit Cat. No. 28704 by QIAGEN
# Transfer cutted gel to a tube.
# Transfer cutted gel to a tube.
-
# Add BufferQC about 3 times as much as the volume of the gel.
+
# Add BufferQG about 3 times as much as the volume of the gel.
-
# Stand the gel for 10 min at 50&#x2103; to Dissolve. If hard to dissolve, sometimes vortex.
+
# Stand the gel for 10min at 50&#x2103; to Dissolve. If hard to dissolve, sometimes vortex.
-
# Confirm the color of the solution. If the color is orange or purple, add about 10&micro;l 3M sodium acetate to yellow.
+
# Confirm the color of the solution. If the color is orange or purple, add about 10&micro;L 3M sodium acetate to yellow.
# Add isopropanol as much as the gel and mix.
# Add isopropanol as much as the gel and mix.
# Apply the solution to the column.
# Apply the solution to the column.
-
# Centrifuge for 1 min at 1300 rpm. Discard the flow-through. If the amount of the sample is much, repeat this step by same column.
+
# Centrifuge for 1min at 1300rpm. Discard the flow-through. If the amount of the sample is much, repeat this step by same column.
-
# Add 500 &micro;l BufferQC and centrifuge for 1 min. Discard the through.
+
# Add 500&micro;L BufferQG and centrifuge for 1min. Discard the through.
-
# Add 750 &micro;l BufferPE and let stand for 2-3 min. If the solution overflows, we decrease the amount of BufferPE.
+
# Add 750&micro;L BufferPE and let stand for 2-3min. If the solution overflows, we decrease the amount of BufferPE.
-
# Centrifuge for 1 min and Discard the through.
+
# Centrifuge for 1min and Discard the through.
-
# Centrifuge for additional 1 min to remove residual buffer.
+
# Centrifuge for additional 1min to remove residual buffer.
# Place the column in a clean tube.
# Place the column in a clean tube.
-
# Add 10 &micro;l BufferEB or H20 to the center of each column, let stand for 1 min, and repeat this step.
+
# Add 10&micro;L BufferEB or MilliQ to the center of each column, let stand for 1min, and repeat this step.
-
# Centrifuge for 1 min at 13000 rpm.
+
# Centrifuge for 1min at 13000rpm.
# Discard the column.
# Discard the column.
-
==Transformation==
+
====Ligation====
-
# Unfreeze conpitent cells on ice.
+
# Make 2&micro;L of Mixture (the vector and the insert at 1 : 5-10) and Control (only the vector).
-
# Dry a plate by letting the plate upside down and partly open in incubator.
+
# Add 5&micro;L Ligation High, 1&micro;L T4 Kinase, and 7&micro;L MilliQ to create a solution.
-
# Add 1 &micro;l DNA solution and 20&micro;l the compitent cells to 1.5ml tube, let stand for 30min on ice.
+
# Incubate at 16&#x2103; for 30 min. If the colonies of E.coli transformed with the Control,
-
#* If few colony is observed, increase the amount of the compitent cells or DNA, but make the amount of DNA not to get over that of the compitent cells.
+
-
# Heatshock for 60s at 42℃.
+
-
# Let stand for 2min on ice.
+
-
# C&micro;lture for 1h in prec&micro;lture medium (LB or SOC medium), and plate by using spreader.
+
-
#* (Do not heat spreader too much because e.coli will dead for heat.
+
-
==Make LB media==
+
====Dephosphorylation====
-
# Wash a graduated cylinder with MilliQ.
+
# Use Bacterial Alkaline Phosphatase (BAP, Takara)
-
# Add the following to about 180ml of MilliQ in the graduated cylinder:
+
# Set the heating block at 65&#x2103;
-
#* Bacto-yeast extract : 1g final to 0.5% (w/v)
+
# Mix the following:
-
#* Trypthon : 2g final to 1% (w/v)
+
#; MilliQ: 24&micro;L
-
#* NaCl : 2g final to 1% (w/v)
+
#; 10x AP Buffer: 5&micro;L
-
#* 200&micro;l 1N NaOH
+
#; DNA sample: 20&micro;L
-
# Seal the graduated cylinder by a Parafilm.
+
#; BAP: 1&micro;L
-
# Dissolve the mixture by inverting the graduated cylinder.
+
#; total: 50&micro;L
-
# Adjust volume to 200ml by adding more MilliQ.
+
#* Not need to purify the DNA sample.
-
# Add the media solution to a 200ml Erlenmeyer flask.
+
# Incubate the solution at 65&#x2103; for 30min.
-
# (To prepare solid media, add 2.4g (1.2%) of agar to the flask.)
+
# Add 50&micro;L MilliQ to the solution (Total is 100&micro;L).
-
# Wrap the tops of the flasks with aluminum foil.
+
# Add 100&micro;L mixed lipid of Phenol and Chloroform (at 1:1).
-
# Place a small piece of auto clave tape on one of the flasks.
+
# Vortex the solution sufficiently.
-
# Autoclave the media on a liquids.
+
# Centrifuge for 5min at 15,000rpm at room temparature and transfer the supernatant to a tube.
-
# Store at room temperature.
+
# Repeat the previous step a few times.
-
# To pour plates, melt the solid media in the microwave, then place flask in water bath to bring media to 50℃.
+
# Add 100&micro;L Chloroform.
-
# Add 200&micro;l of antibiotic:
+
# Vortex the solution.
-
#* 100&micro;g/ml for ampicillin
+
# Centrifuge for 5min at 15,000rpm at room temparature and transfer the supernatant to a tube.
-
#* 50&micro;g/ml for kanamycin
+
# Add 10&micro;L 3M Sodium Acetate and 250&micro;L 100% cold Etanol and mix them.
-
# Add 0.5 - 1.0% Glucose for protein expression.
+
# Let stand for 15min on the ice.
-
;References:
+
# Centrifuge for 15min at 15,000rpm, 4&#x2103; and discard the supernatant.
-
:Bertani, G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62:293-300. PMID 14888646
+
# Add 500&micro;L 70% Etanol.
 +
# Centrifuge for 5min at 15,000rpm, 4&#x2103; and discard the supernatant.
 +
# Dry it.
 +
# Add appropriate quantiles (about 20&micro;L) of TE Buffer to dissolve the DNA.
 +
# Stored at -20&#x2103;.
-
==Make 1% Agarose Gel==
+
====Sequence====
-
# Add the following to a 200ml beaker.
+
# Use Big Dye Terminator 3.1(ABI)
-
#* Agarose : 2g
+
# Mix the following
-
#* 1xTAE : 200ml
+
#; 5xBuffer: 2&micro;L
-
# Wrap the tops of the beaker with plastic wrap.
+
#; Primer  (3.2&micro;M): 1&micro;L
-
# Punch a hole through the wrap with a pipette tip.
+
#; Template Plasmid : 200ng
-
# Dissolve the gel by heating in a microwave and swirling without boiling.
+
#; Big Dye Terminator 3.1: 0.5&micro;L
-
# Pour the gel into the Gel Maker.
+
#; MilliQ: up to 10&micro;L
-
# If there is air bubbles, pushing them with a pipette tip.
+
# Let stand for 1min at 96&#x2103;.
-
# Cover the Gel Maker with a plastic wrap.
+
# 35 cycles for 5s at 98&#x2103;, for 5s 50&#x2103;, and for 2.5min at 68&#x2103;.
-
# Let stand for 45min.
+
# Add 25&micro;L 100% ethanol and 1&micro;L NAOAC
-
# Store in the Tupperware in the refrigerator.
+
-
==BioBrick Assembly==
+
====Ethanol Precipitation====
-
# Restriction Digestion
+
# Use Ethachinmate (NIPPON GENE、312-01791).
-
# Gel Electrophoresis for Separation of Parts from Plasmid Backbone
+
# Add 3.3 &micro;L of 3M Sodium Acetate (attached with Ethachinmate) into 100&micro;L of DNA solution.
-
# Gel Extraction
+
# Add 1&micro;L of Ethachinmate.
-
# Ligation
+
# Vortex.
-
# Transformation
+
# Add ethanol, 200-250&micro;L.
-
# Colony PCR
+
# Vortex. 
-
# Restriction Digestion
+
# Centrifuge at 12000xg for 5min.
-
# Gel Electrophoresis for Confirmation
+
# Precipitation.
-
==Use BioBrick Parts==
+
[[#Top|^Top]]
-
# The Spring 2010 Distribution is sent from iGEM Head Quarter.
+
 
-
# With a pipette tip, punch a hole through the foil cover into the corresponding well to the part that we plan to use.
+
===Measurement===
-
# Add 10&micro;l of milliQ (deionized water).
+
====Promoter Activity (08/24-09/15)====
-
# Transformation.
+
# Pour 5mL the supplemented M9 medium to each test tube.
-
#* (Pipette 1 or 2&micro;l of the resuspended DNA transform into competent cells, plate bacteria with the appropriate antibiotic and grow overnight)
+
# Add 100&micro;L 0.025M IPTG aq to each test tube and mix well.
-
# Pick a single colony and inoc&micro;late broth (again, with the correct antibiotic) and grow for 18 hours.
+
# Pick out a colony on the plate of E.coli and put into the test tube.
-
# Plate C&micro;lture.
+
# Incubate the culture at 37&#x2103; for 16h.
-
# Liquid C&micro;lture.
+
# Measure OD600 of the overnight culture.
-
# Miniprep.
+
# Pour (25/OD600) &micro;L the culture to 1.5mL tube.
-
# Make glycerol stock.
+
# Centrifuge the tube at 14,000rpm and at 4&#x2103; for 2min.
-
# Restriction Digestion.
+
# Discard the supernatant.
-
# Gel Electrophoresis for Confirmation.
+
# Pour 1mL the supplemented M9 medium to the tube and mix well to dissolve pellet.
 +
# Do procedures 7-10 again.
 +
# Pour 25mL the supplemented M9 medium to test tube.
 +
# Add proper amount of 0.025M IPTG aq to the test tube to make the medium including IPTG concentration you wish.
 +
# Add 1mL the medium including IPTG to the pellet in the tube from the test tube and dissolve it.
 +
# Add the all culture in the tube to 24mL the medium in the test tube and make the culture including IPTG concentration you wish. OD600 of this culture is 1.0×10-3.
 +
# Divide 25mL the culture to 5 test tubes by 5mL to measure OD600 and to collect samples of the measurement of GFP fluorescence at 5 different times.
 +
# Incubate these test tubes at 37&#x2103;.
 +
# Measure OD600 of the culture after 4h, 8h, 12h, 16h and 20h.
 +
# Pour 1mL of the culture to 1.5mL tube at the times.
 +
# Centrifuge the 1.5mL tube at 14,000rpm and at 4&#x2103; for 2min.
 +
# Discard the supernatant.
 +
# Freeze the pellets and store it in a fridge as samples for measurement of GFP fluorescence.
 +
# Measure GFP fluorescence.
 +
 
 +
====Promoter Activity (09/16-10/17)====
 +
# Pour 5mL the supplemented M9 medium to each falcon tube.
 +
# Add proper amount of 0.025M IPTG aq to each test tube and mix well to make the medium including IPTG concentration which you hope.
 +
# Pick out a colony on the plate of E.coli and put into three falcon tubes.
 +
# Incubate the culture at 37&#x2103; for 16h.
 +
# Measure OD600 of the overnight culture.
 +
# Pour 5mL the supplemented M9 medium to each new falcon tube. Pre-warm these tubes at 37&#x2103;.
 +
# Pour 50&micro;L the overnight culture to 5mL the fresh medium.
 +
# Incubate the diluted culture at 37&#x2103;.
 +
# Measure OD600 of the culture after 2h, 2.5h, 3h, 3.5h and 4h.
 +
# Pour 1mL of the culture to 1.5mL tube after 3h and 3.5h.
 +
# Centrifuge the 1.5mL tube at 14,000rpm and at 4&#x2103; for 2min.
 +
# Discard the supernatant.
 +
# Freeze the pellets and store it in a fridge as samples for measurement of GFP fluorescence.
 +
# Measure GFP fluorescence.
 +
 
 +
====GFP Fluorescence (from 24 August to 17 October)====
 +
# Add 75&micro;L the cell lysis reagent to the pellet in the 1.5mL tube and mix well and dissolve it.
 +
# Centrifuge the tube at 15,000rpm and at 4&#x2103; for 1min.
 +
# Take out 50&micro;L supernatant and add to each well of a plate.
 +
# Add 50&micro;L the cell lysis reagent to a well to correct the back.
 +
# Set the plate in the plate reader, Wallac 1420 Multilabel Counter, and measure GFP fluorescence of the samples (Ex/Em = 485/535nm, 1sec).
 +
 
 +
====Promoter Activity and GFP Fluorescence (from 18 October)====
 +
# Pour 5mL the supplemented M9 medium to each falcon tube.
 +
# Add proper amount of 0.025M IPTG aq to each test tube and mix well to make the medium including IPTG concentration which you hope.
 +
# Pick out a colony on the plate of E.coli and put into three falcon tubes.
 +
# Incubate the culture at 37&#x2103; for 16h.
 +
# Measure OD600 of the overnight culture.
 +
# Pour 5mL the supplemented M9 medium to each new falcon tube. Pre-warm these tubes at 37&#x2103;.
 +
# Pour 50&micro;L the overnight culture to 5mL the fresh medium.
 +
# Incubate the diluted culture at 37&#x2103;.
 +
# Measure OD600 of the culture after 2h, 2.5h, 3h, 3.5h and 4h.
 +
# Pour 300&micro;L of the culture to 1.5mL tube after 3h and 3.5h and cool it on ice.
 +
# Add 200&micro;L the culture to each well of a plate.
 +
# Add 200&micro;L the supplemented M9 medium to a well to correct tha back.
 +
# Set the plate in the plate reader, Wallac 1420 Multilabel Counter, and measure GFP fluorescence of the samples (Ex/Em = 485/535nm, 1sec).
 +
 
 +
====Quantitative measurement of lytic activity of λ lysis cassette====
 +
# Pour 3mL of supplemented M9 medium to a Falcon tube and add certain amount of IPTG.
 +
# Pick out three colony on the plate of E.coli and put into the Falcon tube.
 +
# Grow it at 30 degreees for 20h.
 +
# Measure OD550 when time of incubation is 16h,18h, and 20h.
 +
# Confirm that the values of OD550 of cultures incubated 16h, 18h, 20h doesn't change.We define it as a parameter of lytic activity, LAV(Lytic avtivity value). 
 +
 
 +
====Measurement of lytic activity with time====
 +
# Pour 3mL of supplemented M9 medium to a Falcon tube.
 +
# Pick out a colony on the plate of E.coli and put into the Falcon tube.
 +
# Grow it at 30 degreees for 16h.
 +
# Dilute it 50 folds with the same media and incubate until OD550 is about 0.15.
 +
# Ditribute 3ml of the culture to falcon tubes and add certain amount of IPTG.
 +
# Incubate the cultures at 30 degrees and measure OD550 of them every 30 min.   
 +
 
 +
 
 +
[[#Top|^Top]]
-
==Ligation==
 
-
# Create a Mixture (the vector and the insert at 1 : 5-10 molec&micro;le) and a Control (only the vector).
 
-
# Add Ligation High to create a solution.
 
-
# Incubate at 16&#x2103; for 30 min.
 
-
* If the colonies of E.coli transformed with the Control,
 
----
----

Latest revision as of 02:42, 28 October 2010

Contents

Protocols

Index

Construction

Solubilization of Antibiotics

  1. Mix the following (Final concentration is 50mg/mL).
    • Ampicillin:
      Ampicillin
      1.0g
      MilliQ
      20mL
    • Kanamycin:
      Kanamycin
      0.5g
      MilliQ
      10mL
  2. Dispense 1.1mL of the solution into 1.5mL tubes.
  3. Store in the -20℃ freezer.

Media

LB Media
  1. Wash a graduated cylinder with MilliQ.
  2. Add the following to about 180mL of MilliQ in the graduated cylinder:
    Bacto-yeast extract
    1.0g (0.5w/v%)
    Tryptone
    2.0g (1.0w/v%)
    NaCl
    2.0g (1.0w/v%)
    1N NaOH
    200µL
  3. Seal the graduated cylinder by a Parafilm.
  4. Dissolve the mixture by inverting the graduated cylinder.
  5. Adjust volume to 200mL by adding more MilliQ.
  6. Add the media solution to a 200mL Erlenmeyer flask.
  7. (To prepare solid media, add 2.4g (1.2w/v%) of agar to the flask.)
  8. Wrap the tops of the flasks with aluminum foil.
  9. Place a small piece of auto clave tape on one of the flasks.
  10. Autoclave the media on liquids.
  11. Store at room temperature.
  12. To pour plates, melt the solid media in the microwave, then place flask in water bath to bring media to 50℃.
  13. Add 200µL of antibiotic
    Ampicillin
    100µg/mL
    Kanamycin
    50µg/mL
  14. (Add 0.5 - 1.0w/v% Glucose for protein expression.)
Supplemented M9 Media
  1. Wash a graduated cylinder with MilliQ.
  2. Add the following to about 180mL of MilliQ in the graduated cylinder:
    Na2HPO4
    1.2g (0.6w/v%)
    KH2PO4
    0.6g (0.3w/v%)
    NaCl
    0.1g (0.05w/v%)
    NH4Cl
    0.2g (0.1w/v%)
  3. Seal the graduated cylinder by a Parafilm.
  4. Dissolve the mixture by inverting the graduated cylinder.
  5. Adjust volume to 200mL by adding more MilliQ.
  6. Add the medium solution to a 200mL Erlenmeyer flask.
  7. Wrap the tops of the flasks with aluminum foil.
  8. Place a small piece of auto clave tape on one of the flasks.
  9. Autoclave the media on a liquids.
  10. Cool at room temperature.
  11. When it become as cool as you can hold it,Add the following to it:
    MgSO4
    1.0mM(final cocentration)
    CaCl2
    0.1mM(final concentration)
    thiamine hydrochloride
    0.001%(final concentration)
    casamino acids
    0.2w/v%
  12. Add 200µL of antibiotic:
    Ampicillin
    100µg/mL
    Kanamycin
    50µg/mL
  13. Add 0.4w/v% Glucose.

Transformation

  1. Unfreeze conpitent cells on ice.
  2. Dry a plate by letting the plate upside down and partly open in incubator.
  3. Add 1µL DNA solution and 20µL compitent cells to 1.5mL tube, let stand for 30min on ice. If few colony is observed, increase the amount of the compitent cells or DNA, but make the amount of DNA not to get over that of the compitent cells.
  4. Heatshock for 60s at 42℃.
  5. Let stand for 2min on ice.
  6. Culture for 1h in preculture medium (LB or SOC medium), and plate by using spreader. Do not heat spreader too much because e.coli will dead for heat.

Miniprep

  1. Use QIAprep Spin Miniprep Kit Cat. No. 27104 by QIAGEN
  2. Pick a single colony from a freshly streaked selective plate and inoculate a culture of about 3mL LB medium containing the appropriate selective antibiotic.
  3. Incubate at 170rpm for 8h at 37℃ with vigorous shaking.
  4. Transfer a half of the culture to a tube.
  5. Harvest the bacterial cells by centrifugation at 14,000g for 1min at 4℃. Remove the medium by decanting.
  6. Transfer the half of the culture to same tube and harvest as same. Remove the medium by pippetting.
  7. Resuspend pelleted bacterial cells in 250µL Buffer P1 and mix thoroughly by pippeting.
  8. Add 250µL Buffer P2 and mix thoroughly by inverting the tube gently 4-6 times.
  9. Add 350µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  10. Centrifuge for 10min at 14,000g at 4℃.
  11. Apply the supernatants from step 10 to the QIAprep spin column by pipetting.
  12. Centrifuge for 10s in a table-top microcentrifuge. Discard the flow-through.
  13. Wash the QIAprep spin column by adding 0.5mL Buffer PB and centrifuging for 10s in a table-top microcentrifuge. Discard the flow-through.
  14. Wash QIAprep spin column by adding 0.65mL Buffer PeE and centrifuging fo 10s in a table-top microcentrifuge.
  15. Discard the flow-through, and centrifuge for and additional 1min to remove residual wash buffer.
  16. Place the QIAprep column in a clean tube. To elute DNA, add 50µL water to the center of each QIAprep spin column, let stand for 1min, and centrifuge for 1min.
  17. Discard the QIAprep spin column.
  18. Measure the concentration of DNA by using eppendorf BioPhotometer plus.
  19. Restriction Digestion.
  20. Agarose Gel Electrophoresis for Confirmation.

Restriction Digestion

  1. Use EcoRI, XbaI, SpeI, PstI, DpnI(NEB)
  2. Mix the following.
    Sample
    5µL
    10xBuffer
    1µL
    Restriction Enzyme
    0.1µL
    MilliQ
    3.9µL
  3. Let stand for 2h at 37℃

PCR

Standard PCR
  1. Dilute template DNA. If the concentration of DNA is 2-100ng/µL, transfer 1µL to a clean tube and add 99µL MilliQ.
  2. Dilute Primer. If the concentration of Primer is XµM, dilute primer X timers and transfer 1µL to a clean tube and add 99µL MilliQ.
  3. Mix the following.
    1. For use of KOD plus ver2:
      25mM MgSO4
      3µL
      2mM dNTPs
      5µL
      10xBuffer for KOD plus ver.2
      5µL
      Template DNA (5ng/µL)
      5µL
      Primer Forward (10µM)
      1.5µL
      Primer Reverse (10µM)
      1.5µL
      KOD plus ver.2
      1µL
      MilliQ
      28µL
      Total
      50µL
    2. For use of KOD FX:
      2mM dNTPs
      10µL
      2xBuffer for KOD FX
      25µL
      Template DNA
      5µL
      Primer Forward (10µM)
      1.5µL
      Primer Reverse (10µM)
      1.5µL
      KOD FX
      1µL
      MilliQ
      6µL
      Total
      50µL
  4. (If amplification is not succeeded, try at 4.5 or 6.0µL 25mM MgSO4.)
  5. Let stand for 2min at 94℃.
  6. 25-40 cycles for 10s at 98℃, for 30s at Tm-5℃, and for 1min (1min for 1kb) at 68℃ (Tm is temparature at which primer will dissolve).
  7. At 15℃ forever.
  8. Agarose Gel Electrophoresis for confirmation.
Screening PCR
  1. Mix the following (Do on PCR Bench).
    10x PCR buffer (TAKARA)
    40µL
    2.5mM dNTP
    8µL
    Primer-1 (10pmol/µL)
    8µL
    Primer-2 (10pmol/µL)
    8µL
    Ex Taq HS (TAKARA)
    1.6µL
    MilliQ 
    334µL (to total 400µL)
  2. Dispense 25µL to 15 tubes.
  3. Pick a single colony and transfer it to each tubes.
  4. Suspend the colony.
  5. Let stand for 10min at 90℃.
  6. 35 cycles for 30s at 94℃, for 30s 55℃, and for 1min at 72℃.
  7. Let stand for 4min at 72℃.
  8. Add 5mL Loading Buffer to the tubes.
  9. Agalose Gel Electrophoresis for confirmation.
  10. Negative Control: Use nothing.
  11. Positive Control: Use a colony that will yield a product with this primers.
Mutagenesis (Point mutation, Deletion, Insertion)
  1. Mix the following.
    10xBuffer
    5µL
    2mM dNTP
    5µL
    Primer Forward (10µM)
    1.5µL
    Primer Reverse (10µM)
    1.5µL
    Template Plasmid (50ng/µL)
    1µL
    KOD plus ver.2
    1µL
    MilliQ
    35µL
    Total
    50µL
  2. Prepare control: instead of KOD plus ver.2, add 1µL MilliQ.
  3. Let stand for 2min at 94℃.
  4. X cycles (1 cycle for 1kb) for 10s at 98℃ and for Ymin (1min for 1kb) at 68℃.
  5. Hold at 4℃.
  6. Take 25µL of the solutions into fresh tubes.
  7. Add 1µL DpnI (10U/µL).
  8. Let stand for 1h at 37℃.
  9. Agarose gel electrophoresis, using 5µL of the solution for confirmation.
  10. Mix the following.
    Sample
    2µL
    Ligation high
    5µL
    T4 Polynucleotide Kinase (5U/µL)
    1µL
    MilliQ
    7µL
    Total
    15µL
  11. Let stand for 1h at 16℃.
  12. Transformation, using 10µL of the solution.
PCR Purification
  1. Use QIAquick PCR Purification Kit Cat. No. 28104 by QIAGEN
  2. Add BufferPB about 5 times as much as the product of PCR.
  3. Apply the solution to the column.
  4. Centrifuge for 1min at 1300rpm. Discard the flow-through. If the amount of the sample is much, repeat this step by same column.
  5. Add 750µL BufferPE and let stand for 2-3min. If the solution overflows, we decrease the amount of BufferPE.
  6. Centrifuge for 1min and discard the through.
  7. Centrifuge for additional 1min to remove residual buffer.
  8. Place the column in a clean tube.
  9. Add 10µL BufferEB or MilliQ to the center of each column, let stand for 1min, and repeat this step.
  10. Centrifuge for 1min at 13000rpm.
  11. Discard the column.

Electrophoresis

  1. Prepare 200mL of a 1.0% agarose solution:
  2. Measure 2.0g agarose into a beaker.
  3. Add 200mL 1xTAE buffer.
  4. Wrap the top of the beaker with plastic wrap.
  5. Punch a hole through the wrap with a pipette tip (To let out steam).
  6. Dissolve the agarose by heating in microwave and swirling without boiling.
  7. Allow the agarose to cool.
  8. Pour the agarose solution into a gel tray on a gel maker.
  9. If there is air bubbles, pushing them with a pipette tip.
  10. Place comb in the maker.
  11. Cover the maker with a plastic wrap.
  12. Let stand for about 45min.
  13. Remove the comb carefully.
  14. Store in the Tupperware in the refrigerator.
  15. Place the tray in electrophoresis chamber.
  16. Cover the tray with 1xTAE buffer.
  17. To prepare samples for electrophoresi, add 1µL of 6x Loading Buffer for every 5µL of DNA solution and mix well.
  18. Load 6µL of the DNA solution per well.
  19. Electrophorese at 100V for about 30min until Loading Buffer have migrated approximately three-quaters of the gel.
  20. Stain the gel in 0.5µg/mL ethidium bromide for 20-30min.
  21. Rinse the gel with MilliQ.
  22. Place a plastic wrap on the transilluminator in the cabinet of Printgraph.
  23. Place the gel on the transilluminator.
  24. Turn on the transilluminator and confirm the position of the gel.
  25. Shoot the picture.
  26. Turn off the transilluminator.
  27. Dispose of the gel.

Gel Extraction

  1. Use QIAquick Gel Extraction Kit Cat. No. 28704 by QIAGEN
  2. Transfer cutted gel to a tube.
  3. Add BufferQG about 3 times as much as the volume of the gel.
  4. Stand the gel for 10min at 50℃ to Dissolve. If hard to dissolve, sometimes vortex.
  5. Confirm the color of the solution. If the color is orange or purple, add about 10µL 3M sodium acetate to yellow.
  6. Add isopropanol as much as the gel and mix.
  7. Apply the solution to the column.
  8. Centrifuge for 1min at 1300rpm. Discard the flow-through. If the amount of the sample is much, repeat this step by same column.
  9. Add 500µL BufferQG and centrifuge for 1min. Discard the through.
  10. Add 750µL BufferPE and let stand for 2-3min. If the solution overflows, we decrease the amount of BufferPE.
  11. Centrifuge for 1min and Discard the through.
  12. Centrifuge for additional 1min to remove residual buffer.
  13. Place the column in a clean tube.
  14. Add 10µL BufferEB or MilliQ to the center of each column, let stand for 1min, and repeat this step.
  15. Centrifuge for 1min at 13000rpm.
  16. Discard the column.

Ligation

  1. Make 2µL of Mixture (the vector and the insert at 1 : 5-10) and Control (only the vector).
  2. Add 5µL Ligation High, 1µL T4 Kinase, and 7µL MilliQ to create a solution.
  3. Incubate at 16℃ for 30 min. If the colonies of E.coli transformed with the Control,

Dephosphorylation

  1. Use Bacterial Alkaline Phosphatase (BAP, Takara)
  2. Set the heating block at 65℃
  3. Mix the following:
    MilliQ
    24µL
    10x AP Buffer
    5µL
    DNA sample
    20µL
    BAP
    1µL
    total
    50µL
    • Not need to purify the DNA sample.
  4. Incubate the solution at 65℃ for 30min.
  5. Add 50µL MilliQ to the solution (Total is 100µL).
  6. Add 100µL mixed lipid of Phenol and Chloroform (at 1:1).
  7. Vortex the solution sufficiently.
  8. Centrifuge for 5min at 15,000rpm at room temparature and transfer the supernatant to a tube.
  9. Repeat the previous step a few times.
  10. Add 100µL Chloroform.
  11. Vortex the solution.
  12. Centrifuge for 5min at 15,000rpm at room temparature and transfer the supernatant to a tube.
  13. Add 10µL 3M Sodium Acetate and 250µL 100% cold Etanol and mix them.
  14. Let stand for 15min on the ice.
  15. Centrifuge for 15min at 15,000rpm, 4℃ and discard the supernatant.
  16. Add 500µL 70% Etanol.
  17. Centrifuge for 5min at 15,000rpm, 4℃ and discard the supernatant.
  18. Dry it.
  19. Add appropriate quantiles (about 20µL) of TE Buffer to dissolve the DNA.
  20. Stored at -20℃.

Sequence

  1. Use Big Dye Terminator 3.1(ABI)
  2. Mix the following
    5xBuffer
    2µL
    Primer (3.2µM)
    1µL
    Template Plasmid 
    200ng
    Big Dye Terminator 3.1
    0.5µL
    MilliQ
    up to 10µL
  3. Let stand for 1min at 96℃.
  4. 35 cycles for 5s at 98℃, for 5s 50℃, and for 2.5min at 68℃.
  5. Add 25µL 100% ethanol and 1µL NAOAC

Ethanol Precipitation

  1. Use Ethachinmate (NIPPON GENE、312-01791).
  2. Add 3.3 µL of 3M Sodium Acetate (attached with Ethachinmate) into 100µL of DNA solution.
  3. Add 1µL of Ethachinmate.
  4. Vortex.
  5. Add ethanol, 200-250µL.
  6. Vortex.
  7. Centrifuge at 12000xg for 5min.
  8. Precipitation.

^Top

Measurement

Promoter Activity (08/24-09/15)

  1. Pour 5mL the supplemented M9 medium to each test tube.
  2. Add 100µL 0.025M IPTG aq to each test tube and mix well.
  3. Pick out a colony on the plate of E.coli and put into the test tube.
  4. Incubate the culture at 37℃ for 16h.
  5. Measure OD600 of the overnight culture.
  6. Pour (25/OD600) µL the culture to 1.5mL tube.
  7. Centrifuge the tube at 14,000rpm and at 4℃ for 2min.
  8. Discard the supernatant.
  9. Pour 1mL the supplemented M9 medium to the tube and mix well to dissolve pellet.
  10. Do procedures 7-10 again.
  11. Pour 25mL the supplemented M9 medium to test tube.
  12. Add proper amount of 0.025M IPTG aq to the test tube to make the medium including IPTG concentration you wish.
  13. Add 1mL the medium including IPTG to the pellet in the tube from the test tube and dissolve it.
  14. Add the all culture in the tube to 24mL the medium in the test tube and make the culture including IPTG concentration you wish. OD600 of this culture is 1.0×10-3.
  15. Divide 25mL the culture to 5 test tubes by 5mL to measure OD600 and to collect samples of the measurement of GFP fluorescence at 5 different times.
  16. Incubate these test tubes at 37℃.
  17. Measure OD600 of the culture after 4h, 8h, 12h, 16h and 20h.
  18. Pour 1mL of the culture to 1.5mL tube at the times.
  19. Centrifuge the 1.5mL tube at 14,000rpm and at 4℃ for 2min.
  20. Discard the supernatant.
  21. Freeze the pellets and store it in a fridge as samples for measurement of GFP fluorescence.
  22. Measure GFP fluorescence.

Promoter Activity (09/16-10/17)

  1. Pour 5mL the supplemented M9 medium to each falcon tube.
  2. Add proper amount of 0.025M IPTG aq to each test tube and mix well to make the medium including IPTG concentration which you hope.
  3. Pick out a colony on the plate of E.coli and put into three falcon tubes.
  4. Incubate the culture at 37℃ for 16h.
  5. Measure OD600 of the overnight culture.
  6. Pour 5mL the supplemented M9 medium to each new falcon tube. Pre-warm these tubes at 37℃.
  7. Pour 50µL the overnight culture to 5mL the fresh medium.
  8. Incubate the diluted culture at 37℃.
  9. Measure OD600 of the culture after 2h, 2.5h, 3h, 3.5h and 4h.
  10. Pour 1mL of the culture to 1.5mL tube after 3h and 3.5h.
  11. Centrifuge the 1.5mL tube at 14,000rpm and at 4℃ for 2min.
  12. Discard the supernatant.
  13. Freeze the pellets and store it in a fridge as samples for measurement of GFP fluorescence.
  14. Measure GFP fluorescence.

GFP Fluorescence (from 24 August to 17 October)

  1. Add 75µL the cell lysis reagent to the pellet in the 1.5mL tube and mix well and dissolve it.
  2. Centrifuge the tube at 15,000rpm and at 4℃ for 1min.
  3. Take out 50µL supernatant and add to each well of a plate.
  4. Add 50µL the cell lysis reagent to a well to correct the back.
  5. Set the plate in the plate reader, Wallac 1420 Multilabel Counter, and measure GFP fluorescence of the samples (Ex/Em = 485/535nm, 1sec).

Promoter Activity and GFP Fluorescence (from 18 October)

  1. Pour 5mL the supplemented M9 medium to each falcon tube.
  2. Add proper amount of 0.025M IPTG aq to each test tube and mix well to make the medium including IPTG concentration which you hope.
  3. Pick out a colony on the plate of E.coli and put into three falcon tubes.
  4. Incubate the culture at 37℃ for 16h.
  5. Measure OD600 of the overnight culture.
  6. Pour 5mL the supplemented M9 medium to each new falcon tube. Pre-warm these tubes at 37℃.
  7. Pour 50µL the overnight culture to 5mL the fresh medium.
  8. Incubate the diluted culture at 37℃.
  9. Measure OD600 of the culture after 2h, 2.5h, 3h, 3.5h and 4h.
  10. Pour 300µL of the culture to 1.5mL tube after 3h and 3.5h and cool it on ice.
  11. Add 200µL the culture to each well of a plate.
  12. Add 200µL the supplemented M9 medium to a well to correct tha back.
  13. Set the plate in the plate reader, Wallac 1420 Multilabel Counter, and measure GFP fluorescence of the samples (Ex/Em = 485/535nm, 1sec).

Quantitative measurement of lytic activity of λ lysis cassette

  1. Pour 3mL of supplemented M9 medium to a Falcon tube and add certain amount of IPTG.
  2. Pick out three colony on the plate of E.coli and put into the Falcon tube.
  3. Grow it at 30 degreees for 20h.
  4. Measure OD550 when time of incubation is 16h,18h, and 20h.
  5. Confirm that the values of OD550 of cultures incubated 16h, 18h, 20h doesn't change.We define it as a parameter of lytic activity, LAV(Lytic avtivity value).

Measurement of lytic activity with time

  1. Pour 3mL of supplemented M9 medium to a Falcon tube.
  2. Pick out a colony on the plate of E.coli and put into the Falcon tube.
  3. Grow it at 30 degreees for 16h.
  4. Dilute it 50 folds with the same media and incubate until OD550 is about 0.15.
  5. Ditribute 3ml of the culture to falcon tubes and add certain amount of IPTG.
  6. Incubate the cultures at 30 degrees and measure OD550 of them every 30 min.


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