Team:Aachen/Notebook/Protocols/Culture medium and conditions

From 2014.igem.org

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= Molecular biological methods =
 
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== Cloning ==
 
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=== Restriction Digest ===
 
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=== Ligation ===
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= Culture medium and culture conditions =
 +
== Media ==
 +
=== LB medium ===
 +
# weight components
 +
#: '''5&nbsp;g/L NaCl'''
 +
#: '''10&nbsp;g/L tryptone'''
 +
#: '''5&nbsp;g/L yeast extract'''
 +
#: (15&nbsp;g/L agar for plates)
 +
# fill up to 1&nbsp;L with deionized water
 +
# '''mix well''' by shaking
 +
# autoclave
 +
## autoclaving tape, caps slightly unscrewed
 +
## base of the pot has to be covered with deionized water
 +
## close lid
 +
## heat '''level 3 until the pressure valve opens'''
 +
## reduce '''heat level to 1.5'''
 +
## set timer to '''20 minutes'''
 +
## turn heater off
 +
## '''wait until the pressure valve retracts''' (30-45 minutes)
 +
## open, close caps & shake
 +
# for plates, wait until you can touch the bottle ('''<60&nbsp;°C''', clean bench!)
 +
# add antibiotics (1&nbsp;µL/mL) and '''shake''' (gloves!)
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=== Gibson Assembly ===
 
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The Gibson Assembly was conducted according to the protocol published by [https://www.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510 New England Biolabs].
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=== TB medium ===
 +
# components 1:
 +
#: '''4&nbsp;mL/L glycerol'''
 +
#: '''12&nbsp;g/L tryptone'''
 +
#: '''24&nbsp;g/L yeast extract'''
 +
# fill up to 900&nbsp;mL with deionized water
 +
# '''mix well''' by shaking
 +
# autoclave
 +
# components 2:
 +
#: '''0.17&nbsp;M KH<sub>2</sub>PO<sub>4</sub>'''
 +
#: '''0.72&nbsp;M K<sub>2</sub>HPO<sub>4</sub>'''
 +
# dissolve in 100&nbsp;mL deionized water and sterilize it by passing it through a filter
 +
# after autoclaving and cooling down, add sterile phosphate solutions
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# Set up the reaction according to the table below on ice (2-3 fragment assembly).
 
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# Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
 
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# Transform NEB 5-alpha Competent E. coli cells with 2 μl of the assembly reaction, following the transformation protocol.
 
 +
=== Hartmans minimal medium (HM) ===
 +
 +
=== M9 minimal medium (M9) ===
<center>
<center>
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{| class="wikitable" style="text-align: right;"
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{| class="wikitable centered"
 +
! '''Components for 1&nbsp;L''' !! '''Volume'''
|-
|-
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| '''Total Amount of Fragments''' || 0.02-0.5&nbsp;pmols
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| bidest. water || style="text-align:right"| 778.667&nbsp;mL
|-
|-
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| '''Gibson Assembly Master Mix (2X)''' || 10&nbsp;µl
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| 10x Salt solution || style="text-align:right"| 100&nbsp;mL
-
|-
+
|-
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| '''Deionized H<sub>2</sub>O''' || 10-X&nbsp;µl
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| Magnesiumsulfatehaptahydrate (10&nbsp;mM) || style="text-align:right"| 100&nbsp;mL
-
|-
+
|-
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| '''Total Volume''' || '''20&nbsp;µl'''
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| Glucose 20% (w/v) || style="text-align:right"| 20&nbsp;mL
 +
|-
 +
| 1000x Trace elements || style="text-align:right"| 1&nbsp;mL
|-
|-
 +
| Thiamin (1&nbsp;mM) || style="text-align:right"| 0.333&nbsp;mL
|}
|}
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</center>
 
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== Transformation ==
 
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=== Heat Shock ===
 
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# thaw cells on ice
 
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# add 1&nbsp;µL of plasmid DNA
 
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# incubate on ice for 30 min
 
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# heat shock at 42&nbsp;°C for 60 s
 
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# incubate on ice for 5 min
 
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# add 200&nbsp;µL of SOC media
 
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# incubate at 37&nbsp;°C for 2&nbsp;h
 
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# plate 20&nbsp; and 200&nbsp;µL on plates supplemented with the appropiate antibiotic
 
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{| class="wikitable centered"
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=== Electroporation ===
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| colspan="3"| '''10x Salt solution'''  
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# add 1&nbsp;μL plasmid to electrocompetent cells
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# put DNA/ cell suspension in electroporation cuvette
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# wipe dry the electroporator
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# use a small plastic pipette to place the cells
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# pulse: 2.5&nbsp;kV, 200-400&nbsp;Ω, 25&nbsp;μF (for ''E.coli'')
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# immediatly add 1&nbsp;mL LB and incubate for 2&nbsp;h at 37&nbsp;°C
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# plate 50&nbsp;μL on selective medium plate
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# centrifuge the rest (3000&nbsp;g, 20 min), discard supernatant, re-suspend the pellet in 50&nbsp;μL LB and plate it on selective medium plate
+
-
 
+
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== PCR ==
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-
 
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We have used several different types of PCR throughout our project:
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-
 
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* colony PCR
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* check PCR
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* gradient PCR
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* SOE PCR
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* touchdown PCR
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* QuikChange(Ligation-During-Amplification)
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-
 
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The scope of appplication as well as the conduct are described below.
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=== Colony PCR /Check PCR ===
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'''With GoTaq Mast Mix'''
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* 12.5 µl GoTaq Master Mix
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* 1 µl primer_F
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* 1 µl primer_R
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* pick colony with tip and suspend in PCR tube
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* 9.5 µl ddH<sub>2</sub>O
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-
 
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<center>
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{| class="wikitable"
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! parameter !! duration !! temp [°C] !!
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|-
|-
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| denature||5:00||95 ||
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! '''Component''' !!''Final concentration''' !! '''Concentration in stock solution'''
|-
|-
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| '''anneal'''||00:30||56 || rowspan="3" | 30 cycles
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| BisTris || style="text-align:right"| 95&nbsp;mM         || style="text-align:right"| 200,000&nbsp;mg/L
|-
|-
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| '''elongate'''||01:00 per kb||72
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| Ammmonium chloride || style="text-align:right"| 60&nbsp;mM || style="text-align:right"| 32,100&nbsp;mg/L
|-
|-
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| '''denature'''||00:30||95
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| Sodium citrate || style="text-align:right"| 12.5&nbsp;mM || style="text-align:right"| 27,000&nbsp;mg/L
|-
|-
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| elongate||05:00||72 || rowspan="2" |
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| Monopotassium phosphate || style="text-align:right"| 3&nbsp;mM || style="text-align:right"| 4,170&nbsp;mg/L
|-
|-
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| store||forever||8
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| Dipotassium phosphate || style="text-align:right"| 0.7&nbsp;mM || style="text-align:right"| 1,590&nbsp;mg/L
 +
|-
|}
|}
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</center>
 
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{| class="wikitable centered"
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=== gradient PCR ===
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| colspan="3"| '''1000x Trace elements'''
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=== SOE PCR ===
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=== touchdown PCR ===
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=== QuikChange ===
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{{Team:Aachen/BlockSeparator}}
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= Analytical methods =
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== Agarose gel electrophoresis==
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Separation of DNA or RNA
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# take 5µl of the PCR product
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# mix with 1µl loading dye
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# apply onto agarose gel together with a marker
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# run at 120°C for 40 minutes for a full gel
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== SDS-PAGE ==
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=== Cell preparation ===
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* lysis cell pellet in lysis buffer
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* centrifuge for 15&nbsp;min at 13.000 rpm
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* mix the supernatant with 2x lammli buffer with β-mercaptoethanol
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* denatured for 5&nbsp;min at 95&nbsp;°C
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* sample to the gel
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For some SDS-PAGEs, we used BioRad ready made gels.
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The recipe of the self-made SDS is as follows:
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=== 1.5x Buffer ===
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* 1.5&nbsp;M Tris-Cl pH = 8.8
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* in 1&nbsp;L is 40&nbsp;ml 10&nbsp;% SDS
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=== Gels ===
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<center>
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{| class="wikitable" style="text-align: right;"
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!
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!! style="border-left: 2px solid #404040;" colspan="3"|0.75&nbsp;mm 12 % RUNNING Gel
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!! style="border-left: 2px solid #404040; background-color:#8ebae5;" colspan="3"|1&nbsp;mm 4 % STACKING Gel
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|-
|-
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|
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! '''Component'''!!'''Final concentration''' !!'''Concentration in stock solution'''
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| style="border-left: 2px solid #404040;"| '''1x''' || '''2x''' || '''4x'''  
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|-
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| style="border-left: 2px solid #404040;"| '''1x''' || '''2x''' || '''4x'''
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| Iron(III) chloride  || style="text-align:right"| 50&nbsp;mM  || style="text-align:right"| 13,515&nbsp;mg/L
 +
|- 
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| Calcium chloride || style="text-align:right"| 20&nbsp;mM || style="text-align:right"| 2,220&nbsp;mg/L
|-
|-
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| '''H{{sub|2}}O'''
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| Manganese(II) chloride      || style="text-align:right"| 10&nbsp;mM || style="text-align:right"| 1,258&nbsp;mg/L
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| style="border-left: 2px solid #404040;"| 1.65&nbsp;mL || 3.3&nbsp;mL || 6.6&nbsp;mL
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|-
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| style="border-left: 2px solid #404040;"| 1.5&nbsp;mL || 3&nbsp;mL || 6&nbsp;mL
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| Zinc sulfate || style="text-align:right"| 10&nbsp;mM || style="text-align:right"| 1,615&nbsp;mg/L
 +
|-
 +
| Cobalt(II) chloride || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 260&nbsp;mg/L
 +
|-
 +
| Copper(II) chloride || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 269&nbsp;mg/L
|-
|-
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| '''1.5x Gel Buffer'''
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| Nickel(II) chloride || style="text-align:right"| 2&nbsp;mM    || style="text-align:right"| 259&nbsp;mg/L
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| style="border-left: 2px solid #404040;"| 1.3&nbsp;mL || 2.6&nbsp;mL || 5.2&nbsp;mL
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|-
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| style="border-left: 2px solid #404040;"| 0.65&nbsp;mL || 1.3&nbsp;mL || 2.6&nbsp;mL
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| Sodium molybdate    || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 412&nbsp;mg/L
|-
|-
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| '''30 % Acrylamide (37.5:1)'''
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| Sodium selenite || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 346&nbsp;mg/L
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| style="border-left: 2px solid #404040;"| 2&nbsp;mL || 4&nbsp;mL || 8&nbsp;mL
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| style="border-left: 2px solid #404040;"| 0.325&nbsp;mL || 0.65&nbsp;mL || 1.3&nbsp;mL
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|-
|-
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| '''10 % APS'''
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| Boric acid || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 124&nbsp;mg/L
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| style="border-left: 2px solid #404040;"| 50&nbsp;µL || 100&nbsp;µL || 200&nbsp;µL
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| style="border-left: 2px solid #404040;"| 25&nbsp;µL || 50&nbsp;µL || 100&nbsp;µL
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|-
|-
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| '''TEMED'''
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| Hydrochloric acid || style="text-align:right"| 1&nbsp;mM || style="text-align:right"| 20&nbsp;mL
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| style="border-left: 2px solid #404040;"| 10&nbsp;µL || 20&nbsp;µL || 40&nbsp;µL
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| style="border-left: 2px solid #404040;"| 5&nbsp;µL || 10&nbsp;µL || 20&nbsp;µL
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|-
|-
|}
|}
</center>
</center>
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==Run gel==
 
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* apply the prepared samples together with a protein marker on the gel
 
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* run the gel for 10&nbsp;min at 60&nbsp;V and after that for ca. 60&nbsp;min at 120&nbsp;V
 
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== Bradford assay ==
 
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Determination of protein concentration
 
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== Measurement of fluorescence ==
 
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The measurement of fluorescence was performed using the Synergy Mx (BioTek) microplate reader and the Gen5 software.
 
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* volume of sample in each well: 100µl
 
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* measure GFP fluorescence at an excitation wavelength of 496&nbsp;nm and an emission wavelength at 516&nbsp;nm
 
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== Measurement of optical density ==
 
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Depending on the number of samples, two different devices were used for measurement of optical density, the Unico Spectrophotometer 1201 (Fisher Bioblock Scientific) and the Synergy Mx (BioTek) microplate reader.
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=== SOC ===
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# components
 +
#: '''0,5&nbsp;% yeast extract'''
 +
#: '''2&nbsp;% tryptone'''
 +
#: '''10&nbsp;mM NaCl'''
 +
#: '''2.5&nbsp;mM KCl'''
 +
#: '''20&nbsp;mM MgSO<sub>4</sub> '''
 +
# fill up with deionized water
 +
# adjust to pH 7.5 with NaOH
 +
# after autoclaving, add 20&nbsp;mM sterile glucose solution (filter sterilization)
{{Team:Aachen/Footer}}
{{Team:Aachen/Footer}}

Revision as of 13:32, 10 October 2014

Culture medium and culture conditions

Media

LB medium

  1. weight components
    5 g/L NaCl
    10 g/L tryptone
    5 g/L yeast extract
    (15 g/L agar for plates)
  2. fill up to 1 L with deionized water
  3. mix well by shaking
  4. autoclave
    1. autoclaving tape, caps slightly unscrewed
    2. base of the pot has to be covered with deionized water
    3. close lid
    4. heat level 3 until the pressure valve opens
    5. reduce heat level to 1.5
    6. set timer to 20 minutes
    7. turn heater off
    8. wait until the pressure valve retracts (30-45 minutes)
    9. open, close caps & shake
  5. for plates, wait until you can touch the bottle (<60 °C, clean bench!)
  6. add antibiotics (1 µL/mL) and shake (gloves!)


TB medium

  1. components 1:
    4 mL/L glycerol
    12 g/L tryptone
    24 g/L yeast extract
  2. fill up to 900 mL with deionized water
  3. mix well by shaking
  4. autoclave
  5. components 2:
    0.17 M KH2PO4
    0.72 M K2HPO4
  6. dissolve in 100 mL deionized water and sterilize it by passing it through a filter
  7. after autoclaving and cooling down, add sterile phosphate solutions


Hartmans minimal medium (HM)

M9 minimal medium (M9)

Components for 1 L Volume
bidest. water 778.667 mL
10x Salt solution 100 mL
Magnesiumsulfatehaptahydrate (10 mM) 100 mL
Glucose 20% (w/v) 20 mL
1000x Trace elements 1 mL
Thiamin (1 mM) 0.333 mL


10x Salt solution
Component' Final concentration Concentration in stock solution
BisTris 95 mM 200,000 mg/L
Ammmonium chloride 60 mM 32,100 mg/L
Sodium citrate 12.5 mM 27,000 mg/L
Monopotassium phosphate 3 mM 4,170 mg/L
Dipotassium phosphate 0.7 mM 1,590 mg/L
1000x Trace elements
ComponentFinal concentration Concentration in stock solution
Iron(III) chloride 50 mM 13,515 mg/L
Calcium chloride 20 mM 2,220 mg/L
Manganese(II) chloride 10 mM 1,258 mg/L
Zinc sulfate 10 mM 1,615 mg/L
Cobalt(II) chloride 2 mM 260 mg/L
Copper(II) chloride 2 mM 269 mg/L
Nickel(II) chloride 2 mM 259 mg/L
Sodium molybdate 2 mM 412 mg/L
Sodium selenite 2 mM 346 mg/L
Boric acid 2 mM 124 mg/L
Hydrochloric acid 1 mM 20 mL


SOC

  1. components
    0,5 % yeast extract
    2 % tryptone
    10 mM NaCl
    2.5 mM KCl
    20 mM MgSO4
  2. fill up with deionized water
  3. adjust to pH 7.5 with NaOH
  4. after autoclaving, add 20 mM sterile glucose solution (filter sterilization)

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