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Lab protocols

On this page you can find a list of important standard protocols we used in our project.

Contents 1 Preparation of chemocompetent DH5-alpha cells 2 Retransformation of BioBricks 3 Transformation of Ligations (in DH5alpha or XL1Blue) 4 Mini-Prep (Promega) 5 Midi-Prep (Promega) 6 Glycerol Stocks (iGEM) 7 Antibiotics Concentration 8 PCR - Clean-Up (Macherey und Nagel) 9 3A - Assembly 10 Strand Directed Mutagenesis PCR 11 Colony PCR

Preparation of chemocompetent DH5-alpha cells

• liquid cultures, OD_600 of 0,6-0,8
• centrifugation at 4 °C, 4500 g, 10 min
• decantation
• resuspend with 40 ml inoune transformation buffer
• repeat centrifugation, decantation and resuspendation
• centrifuge and decantate again
• resuspend in 20 ml inoune transformation buffer
• add 1,5 ml DMSO
• shock freeze in liquid nitrogen

Retransformation of BioBricks

• add 10 µl sterile dest water to DNA on plate
  • incubate for 10 min
  • take 2 µl, leave rest on plate
  • store plates at -20 °C
• add the 2 µl DNA solution to 5 µl competent DH5-alpha
  • incubate for 30 min on ice
  • heat shock for 45 s at 42 °C
  • incubate 3 min on ice
  • add 250 µl LB medium of 37 °C
  • incubate for 45 min at 37 °C, 800 rpm
  • plate 300µl on Agar-plate with appropiate antibiotic
  • dry 15min at room temperatur
  • incubate at 37°C over night

Transformation of Ligations (in DH5alpha or XL1Blue)

• thaw bacteria on ice
• add 2-4µl Ligation mixture to 50µl bacteria
• incubate 30min on ice
• heat shock 30s-45s (XL1Blue preferably 35s) at 42°C
• incubate 6min on ice
• add 250µl prewarmed LB medium (37°C)
• incubate for 45min at 37°C, 800rpm
• plate 300µl on apropiate antibiotic
• dry 15min at room temperature
• incubate at 37°C over night

Mini-Prep (Promega)

• add 1,5 ml overnight-culture in an eppi
• centrifuge for 30 sec, max speed
• decantate
• Repeat previous steps 2-5 times (depending on growth density)
• resuspend with 600 µl dest water
• ad 100 µl cell lysis buffer
• after about 1min (maximum 2min) add 350 µl of neutralization buffer
• centrifuge 3min maximum speed
• put Minicolumn into Collection Tube and transfer supernatant into PureYield^TM Minicolumn
• centrifuge for 15 sec at max speed
• centrifuge for 15 sec at max speed with 200 µl Endotoxin Removal Wash
• centrifuge for 30 sec at max speed with 400 µl Column Wash solution
• put Minicolum into new eppi, abolish liquid
• transfer 30 µl elution buffer into Minicolum, wait for 1 min
• centrifuge for 15 sec at max speed
• store DNA at -20 °C

Midi-Prep (Promega)

• centrifuge 50 ml of liquid cell culture for 10min at 5000g
• decantate
• resuspend with 3 ml resuspension solution
• add 3 ml cell lysis solution and incubate for maximal 3 min at room temperature
• add 5 ml neutralization solution
• centrifuge for 20 min at 20 °C, 5000g
• vacuum pump lysat through cleaning column into binding column
• abolish cleaning column
• vacuum pump with 10 ml endotoxin removal wash solution
• vacuum pump with 20 ml column wash solution
• dry membrane by vacuum
• dd 600 µl nuclease free water on membrane
• centrifuge for 5 min at 1750 g into eppi

Glycerol Stocks (iGEM)

• autoclave glycerol (60%)
• add 1,5 ml cell culture and 0,5 ml Glycerol in a kryo tube
• vortexe
• shock freeze in liquid nitrogen
• store at -80 °C

Antibiotics Concentration

Ampicillin: c=100 mg/ml (dest water) Chloramphenicol: c=18 mg/ml (ethanol) → store at -20 °C

PCR - Clean-Up (Macherey und Nagel)

Gel Extraction:

1. add twice as much NTI volume [µl] than gel weight in mg
2. Incubate 3-7min at 50°C (until gel is dissolved) - shaking at 1000rpm
   • continue with regular Clean-up Protocol

Cleanup:

1. if sample volume < 40µl: fill up with ddH2O to 50µl
2. add twice as much NTI [µl] than sample volume
3. put column into collection tube and transfer solution on the column
4. centrifuge 30s 11.000g and discard flowthrough
5. add 700µl NT3
6. centrifuge 30s 11.000g and discard flowthrough
7. repeat step 5) and 6)
8. centrifuge 1min 11.000g
9. Put column into new eppi - dry at 70°C for 5min
   • small parts (<1000bp):
     1. Put column again into new eppi - add 30µl Elution buffer
     2. Incubate 1min at RT
     3. Centrifuge 1min 11.000g
   • Bigger Parts: (>1000bp)
     1. put Column into new tube - add 20µl Elution buffer
     2. incubate at 70°C for 5min
     3. centrifuge at 50g for 1min
     4. centrifuge at 11.000g for 1min
     5. repeat step 1) to 4)

3A - Assembly

NOTE: Enzymes and buffers were provided by Promega

Restriction (50µl Reaction)

• 25µl Mastermix Restriction-Enzyme Buffer (2x, with BSA)
• 1µl from every restriction enzyme to 500ng backbone equimolar DNA
• fill up to 50µl ddH2O
  • incubate for 1.5h-3h at 37°C
  • inactivate for 20min at 70°C (when directly used for ligation afterwards, without clean-up)

Ligation (20µl Reaction)

• 2,0µl equimolare restriction samples (inserts)
• 1,5µ 20ng backbone
• fill up to 17.7 µl with ddH2O
  • incubate 5min at 37°C
  • add 2µl Ligation buffer (10x) and 0.3µl T4 DNA Ligase
  • incubate for 3h RT or 15°C over night
  • inactivate for 10min at 70°C

Strand Directed Mutagenesis PCR

Mix (50µl reaction)

Substance	Amount
Pfu-buffer (10x)	5µl
MgCl2 (XXX mM)	6µl
dNTP (100mM)	1,5µl
fwd. Primer (100µM)	0,25µl
rev. Primer (100µM)	0,25µl
Pfu	2µl
Template	10ng
ddH2O	fill up to 50µl

PCR-Program:

1. Denaturating 94°C 120s
2. Denaturating 94°C 30s
3. Annealing 55°C 30s
4. Elongation 68°C 720s
5. Repeat step 2) to 4) 11x

Colony PCR

• inoculate 10µl ddH2O with colony

Mix (25µl reaction)

Substance	Amount
Pfu-buffer (10x)	2.5µl
MgCl2 (XXX mM)	3µl
dNTP (100mM)	0.2µl
fwd. Primer (100µM)	0.25µl
rev. Primer (100µM)	0,25µl
Taq	0.32µl
Template	1µl
ddH2O	16.48µl

PCR-Program:

1. Denaturating 94°C 120s
2. Denaturating 94°C 30s
3. Annealing 55°C 30s
4. Elongation 72°C 1min / kb insert
5. Repeat step 2) to 4) 29x
6. final elongation 72°C 180s