Preparation of stock solutions

Making LB media

Dissolve 12.5g of LuriaBroth in 500mL MilliQ H2O and autoclave

Making LB-Agar

Dissolve 12.5g LuriaBroth in 500mL MIlliQ H2O,add 7.5g Agar.
Before use: Heat it inb the microwave and let it cool down to 50 degree celsius before adding Antibiotics.

Antibiotics stock solutions

Ampicillin (amp): 100mg/mL (1000X)
Dissolve 1g Ampicillin in 10mL sterile H2O.Aliquot and store at -20 degree celsius.

Kanamycin (Kan): 50mg/mL (1000X)
Dissolve 0.5g Kanamycin in 10mL sterile H2O.Aliquot and store at -20 degree celsius.

Chloramphenicol(cam): 34mg/mL (1000X)
Dissolve 0.34g Chloramphenicol in 10mL sterile H2O. Aliquot and store at -20 degree celsius.

All stock solutions have to be diluted 1:1000 times when used for Cultures/Plates

Glycerol 20%

Mix 20mL of Glycerole 100% and 80mL of MilliQ H2O, autoclave

Rubidium buffer 1 and 2


Buffer 1
KOAc 30nM
CaCl2 10mM
Glycerol 15%
Adjust pH to 5.8 with Acetic acid
RbCl2 100nM
MnCl2 50nM
Adjust pH to 5.9 with KOH
MIx all components, sterile filter and store at 4 degree celsius

Buffer 2
MOPS 10mM
RbCl2 10mM
CaCl2 75mM
GLycerol 15%
Mix all components and store at 4 degree celsius

Rubidium competent cells


Inoculate 100mL pre-warmed CVM with 2mL of the overnight culture and incubate under shaking (250 rpm) until OD600 : 0.48
Put the culture on ice for 15 minutes and divide it in 2*50 mL falcon tubes
Spin at 4000rpm ,4 degree celsius for 5 minutes
Discard supernatant
Re-suspend pellet in 15mL of ice cold buffer 1
keep on ice for 12 minutes
Spin at 4000rpm ,4 degree celsius for 5 minutes
Carefully resuspend the pellet in 2mL ice cold buffer 2
Aliquot 10*100μL per 50mL culture in 1.5mL tubes and freeze with liquid nitrogen

General cloning procedure

Minipreps

Sigma Aldricht Miniprep kit : Elute in 50μL for higher plasmid concentrations.

Restriction enzyme digest

Figure 1. iGEM suffix insertion

Figure 2. iGEM 3A Assembly

50μL reaction Volume:
2ug of plasmid (max 45μL for low concentration minipreps)
5μL NEB buffer (which buffer for which enzzyme can be looked up on the NEB website)
add ddH2O up to 50μL
0.5/1μL of restriction enzyme (20000U/mL/10000U/mL)
Keep enzymes always on cooling block and don't take them out for too long.
Mix through pipetting

1h at 37 degree celsius
Heat inactivation : 20min at 65 degree celsius(not always necessary)

Dephosphorylation of backbones only (reduces plasmid slef ligation)
Add 1μL of Calf-intestine-phosphatase)
1h at 37 degree celsius
20 minutes heatinactivation at 65 degree celsius.

Add 10μL of loading buffer (6X)
Analysis on gel

Ligation

100ng of backbone and corresponding amount of insert depending on size
Always do a negative control without insert
Add up to 10/15μL with ddH2O
1.1/1.65μL T4 ligation buffer
1μL Ligase
1h at room temperature
20 min at 65 degree celsius

Gel extraction

Sigma Aldricht Gel extraction kit

Transformation

Thaw competent cells on ice for 10 minutes
5μL of ligation product or 1μL of resuspended plasmid/miniprep in 50μL competent cells
30min on ice
1 min heat shock at 42 degree celsius
3 minutes on ice
add 900μL LB
1h shaking at 37 degree celsius
Centrifuge at 12000rcf
remove 750μL supernatant
resuspend the pellet in the remaining 200μL LB
Plate on pre warmed plates

Sequencing

1200ng of DNA
3μL; sequencing primer (10μL)
send out for sequencing using Microsynth account
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PCR (for gene amplification or colony PCR)

PCR was used for gene amplification for cloning and for colony PCR for the clone sleection. For colony PCR TAg Polymerase was used whereas for the gene amplification due too higher needs in accuracy Phusion Polymerase was used. FOr gene Amplification PCR the reaction volume is 50μL and for colony PCR 20uμ. 1-20ng of the template plasmids were used for the amplification. For the coplony PCR in some cases NEB QuickLoad 2X Taq master mix was used.

Experimental procedures

Two layer agar plate experiment

First layer of 1.5% LB-Agar
Second layer consists of 0.7% LB-agar
Receiver cells are dissolevd in 0.7% Agr and then added on top of the first layer
Grow receiver cultures to OD600 of 0.3, dilute into 2mL of 0.7% Agar