Team:Kyoto/Protocols

From 2010.igem.org

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(Promoter Activity (from 16 September to 27 September))
(Measurement the time that cell lysis becomes obvious)
 
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{{:Team:Kyoto/Header}}
{{:Team:Kyoto/Header}}
-
==Index==
+
==Protocols==
-
* [[#Basic|Basic]]
+
===Index===
 +
* [[#Construction|Construction]]
** [[#Solubilization of Antibiotics|Solubilization of Antibiotics]]
** [[#Solubilization of Antibiotics|Solubilization of Antibiotics]]
** [[#Media|Media]]
** [[#Media|Media]]
-
*** [[#LB Media|LB Media]]
 
-
*** [[#Supplemented M9 Media|Supplemented M9 Media]]
 
** [[#Transformation|Transformation]]
** [[#Transformation|Transformation]]
** [[#Miniprep|Miniprep]]
** [[#Miniprep|Miniprep]]
** [[#Restriction Digestion|Restriction Digestion]]
** [[#Restriction Digestion|Restriction Digestion]]
** [[#PCR|PCR]]
** [[#PCR|PCR]]
-
*** [[#Standard PCR|Standard PCR]]
 
-
*** [[#Screening PCR|Screening PCR]]
 
-
*** [[#Mutagenesis (Point mutation, Deletion, Insertion)|Mutagenesis (Point mutation, Deletion, Insertion)]]
 
-
*** [[#PCR Purification|PCR Purification]]
 
** [[#Electrophoresis|Electrophoresis]]
** [[#Electrophoresis|Electrophoresis]]
** [[#Gel Extraction|Gel Extraction]]
** [[#Gel Extraction|Gel Extraction]]
Line 19: Line 14:
** [[#Dephosphorylation|Dephosphorylation]]
** [[#Dephosphorylation|Dephosphorylation]]
** [[#Sequence|Sequence]]
** [[#Sequence|Sequence]]
 +
** [[#Ethanol Precipitation|Ethanol Precipitation]]
* [[#Measurement|Measurement]]
* [[#Measurement|Measurement]]
** [[#Promoter Activity (from 24 August to 15 September)|Promoter Activity (from 24 August to 15 September)]]
** [[#Promoter Activity (from 24 August to 15 September)|Promoter Activity (from 24 August to 15 September)]]
-
** [[#Promoter Activity (from 16 September to 27 September)|Promoter Activity (from 16 September to 27 September)]]
+
** [[#Promoter Activity (from 16 September)|Promoter Activity (from 16 September)]]
-
** [[#GFP Fluorescence|GFP Fluorescence]]
+
** [[#GFP Fluorescence (from 24 August to 17 October)|GFP Fluorescence (from 24 August to 17 October)]]
-
* [[#Lysis|Lysis]]
+
** [[#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]]
** [[#Measurement of the actitvity of inducible lytic system|Measurement of the actitvity of inducible lytic system]]
-
==Basic==
+
===Construction===
-
===Solubilization of Antibiotics===
+
====Solubilization of Antibiotics====
-
# Mix the following (Final concentration is 50mg/ml).
+
# Mix the following (Final concentration is 50mg/mL).
#* Ampicillin:
#* Ampicillin:
#*; Ampicillin: 1.0g
#*; Ampicillin: 1.0g
-
#*; MilliQ: 20ml
+
#*; MilliQ: 20mL
#* Kanamycin:
#* Kanamycin:
#*; Kanamycin: 0.5g
#*; Kanamycin: 0.5g
-
#*; MilliQ: 10ml
+
#*; MilliQ: 10mL
-
# Dispense 1.1ml of the solution into 1.5ml tubes.
+
# Dispense 1.1mL of the solution into 1.5mL tubes.
# Store in the -20℃ freezer.
# Store in the -20℃ freezer.
-
===Media===
+
====Media====
-
====LB Media====
+
=====LB Media=====
# Wash a graduated cylinder with MilliQ.
# Wash a graduated cylinder with MilliQ.
-
# Add the following to about 180ml of MilliQ in the graduated cylinder:
+
# Add the following to about 180mL of MilliQ in the graduated cylinder:
#; Bacto-yeast extract: 1.0g (0.5w/v%)
#; Bacto-yeast extract: 1.0g (0.5w/v%)
#; Tryptone: 2.0g (1.0w/v%)
#; Tryptone: 2.0g (1.0w/v%)
#; NaCl: 2.0g (1.0w/v%)
#; NaCl: 2.0g (1.0w/v%)
-
#; 1N NaOH: 200µl
+
#; 1N NaOH: 200µL
# Seal the graduated cylinder by a Parafilm.
# Seal the graduated cylinder by a Parafilm.
# Dissolve the mixture by inverting the graduated cylinder.
# Dissolve the mixture by inverting the graduated cylinder.
-
# Adjust volume to 200ml by adding more MilliQ.
+
# Adjust volume to 200mL by adding more MilliQ.
-
# Add the media solution to a 200ml Erlenmeyer flask.
+
# Add the media solution to a 200mL Erlenmeyer flask.
# (To prepare solid media, add 2.4g (1.2w/v%) of agar to the 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.
# Wrap the tops of the flasks with aluminum foil.
# Place a small piece of auto clave tape on one of the flasks.
# Place a small piece of auto clave tape on one of the flasks.
-
# Autoclave the media on a liquids.
+
# Autoclave the media on liquids.
# Store at room temperature.
# 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℃.
+
# 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
+
# Add 200µL of antibiotic
-
#; Ampicillin: 100µg/ml
+
#; Ampicillin: 100µg/mL
-
#; Kanamycin: 50µg/ml
+
#; Kanamycin: 50µg/mL
# (Add 0.5 - 1.0w/v% Glucose for protein expression.)
# (Add 0.5 - 1.0w/v% Glucose for protein expression.)
-
====Supplemented M9 Media====
+
=====Supplemented M9 Media=====
# Wash a graduated cylinder with MilliQ.
# Wash a graduated cylinder with MilliQ.
-
# Add the following to about 180ml of MilliQ in the graduated cylinder:
+
# Add the following to about 180mL of MilliQ in the graduated cylinder:
-
#; Na2HPO4: 1.2g(0.6w/v%)
+
#; Na2HPO4: 1.2g (0.6w/v%)
-
#; KH2PO4: 0.6g(0.3w/v%)
+
#; KH2PO4: 0.6g (0.3w/v%)
-
#; NaCl: 0.1g(0.05w/v%)
+
#; NaCl: 0.1g (0.05w/v%)
-
#; NH4Cl: 0.2g(0.1w/v%)
+
#; NH4Cl: 0.2g (0.1w/v%)
# Seal the graduated cylinder by a Parafilm.  
# Seal the graduated cylinder by a Parafilm.  
# Dissolve the mixture by inverting the graduated cylinder.
# Dissolve the mixture by inverting the graduated cylinder.
-
# Adjust volume to 200ml by adding more MilliQ.
+
# Adjust volume to 200mL by adding more MilliQ.
-
# Add the medium solution to a 200ml Erlenmeyer flask.
+
# Add the medium solution to a 200mL Erlenmeyer flask.
# Wrap the tops of the flasks with aluminum foil.
# Wrap the tops of the flasks with aluminum foil.
# Place a small piece of auto clave tape on one of the flasks.
# Place a small piece of auto clave tape on one of the flasks.
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#; thiamine hydrochloride: 0.001%(final concentration)
#; thiamine hydrochloride: 0.001%(final concentration)
#; casamino acids: 0.2w/v%
#; casamino acids: 0.2w/v%
-
# Add 200µl of antibiotic:  
+
# Add 200µL of antibiotic:  
-
#; Ampicillin: 100µg/ml
+
#; Ampicillin: 100µg/mL
-
#; Kanamycin: 50µg/ml
+
#; Kanamycin: 50µg/mL
# Add 0.4w/v% Glucose.
# Add 0.4w/v% Glucose.
-
===Transformation===
+
====Transformation====
# Unfreeze conpitent cells on ice.
# Unfreeze conpitent cells on ice.
# Dry a plate by letting the plate upside down and partly open in incubator.
# 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.
+
# 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℃.
# Heatshock for 60s at 42℃.
# Let stand for 2min on ice.
# 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.
# 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===
+
====Miniprep====
# Use QIAprep Spin Miniprep Kit Cat. No. 27104 by QIAGEN
# 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.
+
# 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.
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# Agarose Gel Electrophoresis for Confirmation.
# Agarose Gel Electrophoresis for Confirmation.
-
===Restriction Digestion===
+
====Restriction Digestion====
-
# Use EcoRI, XbaI, SpeI, PstI(NEB)
+
# Use EcoRI, XbaI, SpeI, PstI, DpnI(NEB)
# Mix the following.
# Mix the following.
-
## Sample
+
#; Sample: 5µL
-
## 10xBuffer
+
#; 10xBuffer: 1µL
-
## MilliQ  
+
#; Restriction Enzyme: 0.1µL
-
## Restriction Enzyme
+
#; MilliQ: 3.9µL
# Let stand for 2h at 37℃
# Let stand for 2h at 37℃
-
===PCR===
+
====PCR====
-
====Standard 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 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 Xµmol/l, dilute primer X timers and 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.
# Mix the following.
## For use of KOD plus ver2:
## For use of KOD plus ver2:
-
##; 25mmol/l MgSO4: 3µl
+
##; 25mM MgSO4: 3µL
-
##; 2mmol/l dNTPs: 5µl
+
##; 2mM dNTPs: 5µL
-
##; 10xBuffer for KOD plus ver.2: 5µl
+
##; 10xBuffer for KOD plus ver.2: 5µL
-
##; Template DNA  (5ng/µl): 5µl
+
##; Template DNA  (5ng/µL): 5µL
-
##; Primer Forward (10µmol/l): 1.5µl
+
##; Primer Forward (10µM): 1.5µL
-
##; Primer Reverse (10µmol/l): 1.5µl
+
##; Primer Reverse (10µM): 1.5µL
-
##; KOD plus ver.2: 1µl
+
##; KOD plus ver.2: 1µL
-
##; MilliQ: 28µl
+
##; MilliQ: 28µL
-
##; Total: 50µl
+
##; Total: 50µL
## For use of KOD FX:
## For use of KOD FX:
-
##; 2mmol/l dNTPs: 10µl
+
##; 2mM dNTPs: 10µL
-
##; 2xBuffer for KOD FX: 25µl
+
##; 2xBuffer for KOD FX: 25µL
-
##; Template DNA: 5µl
+
##; Template DNA: 5µL
-
##; Primer Forward (10µmol/l): 1.5µl
+
##; Primer Forward (10µM): 1.5µL
-
##; Primer Reverse (10µmol/l): 1.5µl
+
##; Primer Reverse (10µM): 1.5µL
-
##; KOD FX: 1µl
+
##; KOD FX: 1µL
-
##; MilliQ: 6µl
+
##; MilliQ: 6µL
-
##; Total: 50µl
+
##; Total: 50µL
-
# (If amplification is not succeeded, try at 4.5 or 6.0µl 25mmol/l MgSO4.)
+
# (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 155: Line 151:
# Agarose Gel Electrophoresis for confirmation.
# Agarose Gel Electrophoresis for confirmation.
-
====Screening PCR====
+
=====Screening PCR=====
# Mix the following (Do on PCR Bench).
# Mix the following (Do on PCR Bench).
-
#; 10x PCR buffer (TAKARA): 40µl
+
#; 10x PCR buffer (TAKARA): 40µL
-
#; 2.5mmol/l dNTP: 8µl
+
#; 2.5mM dNTP: 8µL
-
#; Primer-1 (10pmol/µl): 8µl
+
#; Primer-1 (10pmol/µL): 8µL
-
#; Primer-2 (10pmol/µl): 8µl
+
#; Primer-2 (10pmol/µL): 8µL
-
#; Ex Taq HS (TAKARA): 1.6µl
+
#; Ex Taq HS (TAKARA): 1.6µL
-
#; MilliQ : 334µl (to total 400µl)
+
#; MilliQ : 334µL (to total 400µL)
-
# Dispense 25µl to 15 tubes.
+
# Dispense 25µL to 15 tubes.
# Pick a single colony and transfer it to each tubes.
# Pick a single colony and transfer it to each tubes.
# Suspend the colony.
# Suspend the colony.
-
# Let stand for 10min at 90℃.
+
# Let stand for 10min at 90℃.
-
# 35 cycles for 30s at 94℃, for 30s 55℃, and for 1min at 72℃.
+
# 35 cycles for 30s at 94℃, for 30s 55℃, and for 1min at 72℃.
-
# Let stand for 4min at 72℃.  
+
# Let stand for 4min at 72℃.  
-
# Add 5ml Loading Buffer to the tubes.
+
# Add 5mL Loading Buffer to the tubes.
# Agalose Gel Electrophoresis for confirmation.
# Agalose Gel Electrophoresis for confirmation.
# Negative Control: Use nothing.
# Negative Control: Use nothing.
# Positive Control: Use a colony that will yield a product with this primers.
# Positive Control: Use a colony that will yield a product with this primers.
-
====Mutagenesis (Point mutation, Deletion, Insertion)====
+
=====Mutagenesis (Point mutation, Deletion, Insertion)=====
# Mix the following.
# Mix the following.
-
#; 10xBuffer: 5µl
+
#; 10xBuffer: 5µL
-
#; 2mmol/l dNTP: 5µl
+
#; 2mM dNTP: 5µL
-
#; Primer Forward (10pmol/µl): 1.5µl
+
#; Primer Forward (10µM): 1.5µL
-
#; Primer Reverse (10pmol/µl): 1.5µl
+
#; Primer Reverse (10µM): 1.5µL
-
#; Template Plasmid (50ng/µl): 1µl
+
#; Template Plasmid (50ng/µL): 1µL
-
#; KOD plus ver.2: 1µl
+
#; KOD plus ver.2: 1µL
-
#; MilliQ: 35µl
+
#; MilliQ: 35µL
-
#; Total: 50µl
+
#; Total: 50µL
-
# Prepare control: instead of KOD plus ver.2, add 1µl MilliQ.
+
# Prepare control: instead of KOD plus ver.2, add 1µL MilliQ.
# Let stand for 2min at 94℃.  
# Let stand for 2min at 94℃.  
-
# Xcycles (1cycle for 1kb) for 10s at 98℃ and for Ymin (1min for 1kb) at 68℃.
+
# X cycles (1 cycle for 1kb) for 10s at 98℃ and for Ymin (1min for 1kb) at 68℃.
# Hold at 4℃.
# Hold at 4℃.
-
# Take 25µl of the solutions into fresh tubes.
+
# Take 25µL of the solutions into fresh tubes.
-
# Add 1µl ''Dpn''I (10U/µl).
+
# Add 1µL ''Dpn''I (10U/µL).
# Let stand for 1h at 37℃.
# Let stand for 1h at 37℃.
-
# Agarose gel electrophoresis, using 5µl of the solution for confirmation.
+
# Agarose gel electrophoresis, using 5µL of the solution for confirmation.
# Mix the following.
# Mix the following.
-
#; Sample: 2µl
+
#; Sample: 2µL
-
#; Ligation high: 5µl
+
#; Ligation high: 5µL
-
#; T4 Polynucleotide Kinase (5U/µl): 1µl
+
#; T4 Polynucleotide Kinase (5U/µL): 1µL
-
#; MilliQ: 7µl
+
#; MilliQ: 7µL
-
#; Total: 15µl
+
#; Total: 15µL
# Let stand for 1h at 16℃.
# Let stand for 1h at 16℃.
-
# Transformation, using 10µl of the solution.
+
# Transformation, using 10µL of the solution.
-
====PCR Purification====
+
=====PCR Purification=====
# Use QIAquick PCR Purification Kit Cat. No. 28104 by QIAGEN  
# Use QIAquick PCR Purification Kit Cat. No. 28104 by QIAGEN  
# Add BufferPB about 5 times as much as the product of PCR.
# Add BufferPB about 5 times as much as the product of PCR.
# 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 750 µl BufferPE and let stand for 2-3 min. If the solution overflows, we decrease the amount of BufferPE.
+
# Add 750µL 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 µl BufferEB or H20 to the center of each column, let stand for 1 min, and repeat this step.
+
# Add 10µL 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.
-
===Electrophoresis===
+
====Electrophoresis====
-
# Prepare 200ml of a 1.0% agarose solution:
+
# Prepare 200mL of a 1.0% agarose solution:
# Measure 2.0g agarose into a beaker.
# Measure 2.0g agarose into a beaker.
-
# Add 200ml 1xTAE buffer.
+
# Add 200mL 1xTAE buffer.
# Wrap the top of the beaker with plastic wrap.
# Wrap the top of the beaker with plastic wrap.
# Punch a hole through the wrap with a pipette tip (To let out steam).
# Punch a hole through the wrap with a pipette tip (To let out steam).
Line 231: Line 227:
# Place the tray in electrophoresis chamber.
# Place the tray in electrophoresis chamber.
# Cover the tray with 1xTAE buffer.
# Cover the tray with 1xTAE buffer.
-
# To prepare samples for electrophoresi, add 1µl of 6x Loading Buffer for every 5µl of DNA solution and mix well.
+
# To prepare samples for electrophoresi, add 1µL of 6x Loading Buffer for every 5µL of DNA solution and mix well.
-
# Load 6µl of the DNA solution per well.
+
# Load 6µL of the DNA solution per well.
-
# Electrophorese at 100volts for about 30min until Loading Buffer have migrated approximately three-quaters of the gel.
+
# Electrophorese at 100V for about 30min until Loading Buffer have migrated approximately three-quaters of the gel.
-
# Stain the gel in 0.5µg/ml ethidium bromide for 20-30min.
+
# Stain the gel in 0.5µg/mL ethidium bromide for 20-30min.
# Rinse the gel with MilliQ.
# Rinse the gel with MilliQ.
# Place a plastic wrap on the transilluminator in the cabinet of Printgraph.
# Place a plastic wrap on the transilluminator in the cabinet of Printgraph.
Line 243: Line 239:
# Dispose of the gel.
# Dispose of the gel.
-
===Gel Extraction===
+
====Gel Extraction====
# Use QIAquick Gel Extraction Kit Cat. No. 28704 by QIAGEN  
# Use QIAquick Gel Extraction Kit Cat. No. 28704 by QIAGEN  
# Transfer cutted gel to a tube.
# Transfer cutted gel to a tube.
# Add BufferQG 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℃ to Dissolve. If hard to dissolve, sometimes vortex.
+
# Stand the gel for 10min at 50℃ to Dissolve. If hard to dissolve, sometimes vortex.
-
# Confirm the color of the solution. If the color is orange or purple, add about 10µl 3M sodium acetate to yellow.
+
# Confirm the color of the solution. If the color is orange or purple, add about 10µL 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 µl BufferQG and centrifuge for 1 min. Discard the through.
+
# Add 500µL BufferQG and centrifuge for 1min. Discard the through.
-
# Add 750 µl BufferPE and let stand for 2-3 min. If the solution overflows, we decrease the amount of BufferPE.
+
# Add 750µL 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 µl BufferEB or H20 to the center of each column, let stand for 1 min, and repeat this step.
+
# Add 10µL 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.
-
===Ligation===
+
====Ligation====
-
# Create a Mixture (the vector and the insert at 1 : 5-10 molecµle) and a Control (only the vector).
+
# Make 2µL of Mixture (the vector and the insert at 1 : 5-10) and Control (only the vector).
-
# Add Ligation High to create a solution.
+
# Add 5µL Ligation High, 1µL T4 Kinase, and 7µL MilliQ to create a solution.
# Incubate at 16℃ for 30 min. If the colonies of E.coli transformed with the Control,  
# Incubate at 16℃ for 30 min. If the colonies of E.coli transformed with the Control,  
-
===Dephosphorylation===
+
====Dephosphorylation====
# Use Bacterial Alkaline Phosphatase (BAP, Takara)
# Use Bacterial Alkaline Phosphatase (BAP, Takara)
# Set the heating block at 65℃
# Set the heating block at 65℃
# Mix the following:
# Mix the following:
-
#; MilliQ: 24µl
+
#; MilliQ: 24µL
-
#; 10x AP Buffer: 5µl
+
#; 10x AP Buffer: 5µL
-
#; DNA sample: 20µl
+
#; DNA sample: 20µL
-
#; BAP: 1µl
+
#; BAP: 1µL
-
#; total: 50µl
+
#; total: 50µL
#* Not need to purify the DNA sample.
#* Not need to purify the DNA sample.
# Incubate the solution at 65℃ for 30min.
# Incubate the solution at 65℃ for 30min.
-
# Add 50µl MilliQ to the solution (Total is 100µl).
+
# Add 50µL MilliQ to the solution (Total is 100µL).
-
# Add 100µl mixed lipid of Phenol and Chloroform (1:1).
+
# Add 100µL mixed lipid of Phenol and Chloroform (at 1:1).
# Vortex the solution sufficiently.
# Vortex the solution sufficiently.
# Centrifuge for 5min at 15,000rpm at room temparature and transfer the supernatant to a tube.
# Centrifuge for 5min at 15,000rpm at room temparature and transfer the supernatant to a tube.
# Repeat the previous step a few times.
# Repeat the previous step a few times.
-
# Add 100µl Chloroform.
+
# Add 100µL Chloroform.
# Vortex the solution.
# Vortex the solution.
# Centrifuge for 5min at 15,000rpm at room temparature and transfer the supernatant to a tube.
# Centrifuge for 5min at 15,000rpm at room temparature and transfer the supernatant to a tube.
-
# Add 10µl 3mol/l Sodium Acetate and 250µl 100% cold Etanol and mix them.
+
# Add 10µL 3M Sodium Acetate and 250µL 100% cold Etanol and mix them.
# Let stand for 15min on the ice.
# Let stand for 15min on the ice.
# Centrifuge for 15min at 15,000rpm, 4℃ and discard the supernatant.
# Centrifuge for 15min at 15,000rpm, 4℃ and discard the supernatant.
-
# Add 500µl 70% Etanol.
+
# Add 500µL 70% Etanol.
# Centrifuge for 5min at 15,000rpm, 4℃ and discard the supernatant.
# Centrifuge for 5min at 15,000rpm, 4℃ and discard the supernatant.
# Dry it.
# Dry it.
-
# Add appropriate quantiles (about 20µl) of TE Buffer to dissolve the DNA.
+
# Add appropriate quantiles (about 20µL) of TE Buffer to dissolve the DNA.
# Stored at -20℃.
# Stored at -20℃.
-
===Sequence===
+
====Sequence====
# Use Big Dye Terminator 3.1(ABI)
# Use Big Dye Terminator 3.1(ABI)
# Mix the following
# Mix the following
-
#; 5xBuffer: 2µl
+
#; 5xBuffer: 2µL
-
#; Primer  (3.2pmol/µl): 1µl
+
#; Primer  (3.2µM): 1µL
#; Template Plasmid : 200ng
#; Template Plasmid : 200ng
-
#; Big Dye Terminator 3.1: 0.5µl
+
#; Big Dye Terminator 3.1: 0.5µL
-
#; MilliQ: up to 10µl
+
#; MilliQ: up to 10µL
-
# Let stand for 1min at 96℃.
+
# Let stand for 1min at 96℃.
-
# 35 cycles for 5s at 98℃, for 5s 50℃, and for 2.5min at 68℃.
+
# 35 cycles for 5s at 98℃, for 5s 50℃, and for 2.5min at 68℃.
-
# Add 25µl 100% ethanol and 1ul NAOAC
+
# Add 25µL 100% ethanol and 1µL NAOAC
-
===Ethanol Precipitation===
+
====Ethanol Precipitation====
-
Use Ethachinmate(NIPPON GENE、312-01791)
+
# Use Ethachinmate (NIPPON GENE、312-01791).
-
# Add 3.3 μl of 3mol/l Sodium Acetate (attached with Ethachinmate) into 100µl of DNA solution.  
+
# Add 3.3 µL of 3M Sodium Acetate (attached with Ethachinmate) into 100µL of DNA solution.
-
# Add 1µl of Ethachinmate
+
# Add 1µL of Ethachinmate.
-
# Vortex
+
# Vortex.
-
# Add ethanol, 200~250µl
+
# Add ethanol, 200-250µL.
# Vortex.   
# Vortex.   
-
# Centrifuge at 12000 x g for 5 minutes
+
# Centrifuge at 12000xg for 5min.
-
# Precipitation
+
# Precipitation.
-
==Measurement==
+
[[#Top|^Top]]
-
===Promoter Activity (from 24 August to 15 September)===
+
 
 +
===Measurement===
 +
====Promoter Activity (08/24-09/15)====
# Pour 5mL the supplemented M9 medium to each test tube.
# Pour 5mL the supplemented M9 medium to each test tube.
-
# Add 100μL 0.025M IPTG aq to each test tube and mix well.
+
# Add 100µL 0.025M IPTG aq to each test tube and mix well.
# Pick out a colony on the plate of E.coli and put into the test tube.
# Pick out a colony on the plate of E.coli and put into the test tube.
-
# Incubate the culture at 37℃ for 16h.
+
# Incubate the culture at 37℃ for 16h.
# Measure OD600 of the overnight culture.
# Measure OD600 of the overnight culture.
-
# Pour (25/OD600)μL the culture to 1.5mL tube.
+
# Pour (25/OD600) µL the culture to 1.5mL tube.
-
# Centrifuge the tube at 14,000rpm and at 4℃ for 2minutes.
+
# Centrifuge the tube at 14,000rpm and at 4℃ for 2min.
# Discard the supernatant.
# Discard the supernatant.
# Pour 1mL the supplemented M9 medium to the tube and mix well to dissolve pellet.
# Pour 1mL the supplemented M9 medium to the tube and mix well to dissolve pellet.
-
# Do procedures 7 and 8 again.
+
# Do procedures 7-10 again.
-
# Do procedures 9 and 10 again.
+
# Pour 25mL the supplemented M9 medium to test tube.
# 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 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 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.
+
# 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.
# 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℃.
+
# Incubate these test tubes at 37℃.
# Measure OD600 of the culture after 4h, 8h, 12h, 16h and 20h.
# Measure OD600 of the culture after 4h, 8h, 12h, 16h and 20h.
# Pour 1mL of the culture to 1.5mL tube at the times.
# Pour 1mL of the culture to 1.5mL tube at the times.
-
# Centrifuge the 1.5mL tube at 14,000rpm and at 4℃ for 2minutes.
+
# Centrifuge the 1.5mL tube at 14,000rpm and at 4℃ for 2min.
# Discard the supernatant.
# Discard the supernatant.
# Freeze the pellets and store it in a fridge as samples for measurement of GFP fluorescence.
# Freeze the pellets and store it in a fridge as samples for measurement of GFP fluorescence.
# Measure GFP fluorescence.
# Measure GFP fluorescence.
-
To know details, see “how to measure GFP fluorescence”.
 
-
===Promoter Activity (from 16 September)===
+
====Promoter Activity (09/16-10/17)====
# Pour 5mL the supplemented M9 medium to each falcon tube.
# 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.
# 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.
# Pick out a colony on the plate of E.coli and put into three falcon tubes.
-
# Incubate the culture at 37℃ for 16h.
+
# Incubate the culture at 37℃ for 16h.
# Measure OD600 of the overnight culture.
# Measure OD600 of the overnight culture.
-
# Pour 5mL the supplemented M9 medium to each new falcon tube. Pre-warm these tubes at 37℃.
+
# Pour 5mL the supplemented M9 medium to each new falcon tube. Pre-warm these tubes at 37℃.
-
# Pour 50μL the overnight culture to 5mL the fresh medium.
+
# Pour 50µL the overnight culture to 5mL the fresh medium.
-
# Incubate the diluted culture at 37℃.
+
# Incubate the diluted culture at 37℃.
# Measure OD600 of the culture after 2h, 2.5h, 3h, 3.5h and 4h.
# 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.
# 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℃ for 2minutes.
+
# Centrifuge the 1.5mL tube at 14,000rpm and at 4℃ for 2min.
# Discard the supernatant.
# Discard the supernatant.
# Freeze the pellets and store it in a fridge as samples for measurement of GFP fluorescence.
# Freeze the pellets and store it in a fridge as samples for measurement of GFP fluorescence.
# Measure GFP fluorescence.
# Measure GFP fluorescence.
-
 
-
To know details, see “how to measure GFP fluorescence”.
 
-
===GFP Fluorescence===
+
====GFP Fluorescence (from 24 August to 17 October)====
-
# Add 75μL the cell lysis reagent to the pellet in the 1.5mL tube and mix well and dissolve it.
+
# Add 75µL 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℃ for 1minutes.
+
# Centrifuge the tube at 15,000rpm and at 4℃ for 1min.
-
# Take out 50μL supernatant and add to each well of a plate.
+
# Take out 50µL supernatant and add to each well of a plate.
-
# Add 50μL the cell lysis reagent to a well to correct the back.
+
# Add 50µL 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).
# Set the plate in the plate reader, Wallac 1420 Multilabel Counter, and measure GFP fluorescence of the samples (Ex/Em = 485/535nm, 1sec).
-
==Lysis==
+
====Promoter Activity and GFP Fluorescence (from 18 October)====
-
===Measurement of lytic system===
+
# Pour 5mL the supplemented M9 medium to each falcon tube.
-
# Pour 3mL of supplemented M9 medium to a 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 the falcon tube.
+
# Pick out a colony on the plate of E.coli and put into three falcon tubes.
-
# Grow it at 37℃ for 16h.
+
# Incubate the culture at 37℃ for 16h.
-
# Measure A550 of the overnight culture.
+
# Measure OD600 of the overnight culture.
-
# Dilute it with pre-warmed supplemented M9 medium to A550 be 0.20.
+
# Pour 5mL the supplemented M9 medium to each new falcon tube. Pre-warm these tubes at 37℃.
-
# Pour 3mL of the adjusted culture to a falcon tube.
+
# Pour 50µL the overnight culture to 5mL the fresh medium.
-
# Add proper amount of IPTG.
+
# Incubate the diluted culture at 37℃.
-
# Grow the culture at 37℃.
+
# Measure OD600 of the culture after 2h, 2.5h, 3h, 3.5h and 4h.
-
# Measure A550 of the culture every 30 min until 4h.
+
# Pour 300µL of the culture to 1.5mL tube after 3h and 3.5h and cool it on ice.
 +
# Add 200µL the culture to each well of a plate.
 +
# Add 200µL 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]]
 +
 
 +
 
 +
 
----
----

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.

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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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